Pyrazole pyrimidine derivative and uses thereof

ABSTRACT

The present invention provides pyrazole pyrimidine derivatives which inhibit Casein kinase I (CKI) and/or Interleukin-1 receptor-associated kinase 1 (IRAKI) and methods of their manufacture, compositions comprising them and uses thereof in methods of treating malignant disease and disorders and methods for treating inflammatory diseases and disorders.

TECHNOLOGICAL FIELD

The present invention provides pyrazole pyrimidine derivatives and usesthereof in methods of treating malignant disease and disorders andmethods for treating inflammatory diseases and disorders.

BACKGROUND

The casein kinase 1 family (CK1, or CKI) are serine/threonine kinaseswith six members (isoforms) in humans: α, γ1, γ2, γ3, δ and ε. Theydiffer in length and sequence of the N-terminal (9-76 amino acids) andespecially the C-terminal (24-200 amino acids) non-catalytic domain(Schittek and Sinnberg, Molecular Cancer 2014, 13:231).

CK1δ and CK1ε are 98% identical in their kinase domain and 53% identicalin their C-terminal regulatory domain (Fish K J et al. J Biol Chem 1995,270:14875-14883). Whereas, there is some redundancy with respect to CK1substrate phosphorylation, most CK1 isoforms have distinct biologicalroles. The wide range of CK1 substrates shows that the CK1 familymembers are involved in multiple cellular processes, from regulation ofmembrane trafficking, cytokinesis, vesicular transport, ribosomebiogenesis, DNA repair, signal transduction pathways, apoptosis and inthe circadian rhythm (Knippschild U et al. Cell Signal 2005, 17:675-689;Cheong J K and Virshup D M. Int J Biochem Cell Biol 2011, 43:465-469;Zemp I, et al. J Cell Sci 2014, 127:1242-1253).

CK1α plays a role in the mitotic spindle formation during cell divisionand in DNA repair mechanisms and participates in RNA metabolism(Knippschild U et al. Cell Signal 2005, 17:675-689). It contributes tothe activation of mTOR via sustained degradation of the endogenous mTORinhibitor DEPTOR (Duan S et al. Mol Cell 2011, 44:317-324).

CK1α has a major role in regulation of the Wnt/β-catenin signalingpathway. The inventors of this application have shown that CK1α is a keycomponent of the β-catenin destruction complex. When the Wnt receptorsare not engaged, CK1α phosphorylates β-catenin at serine residue S45,which is necessary for the priming phosphorylation of another kinase,GSK3 (Amit et al. Genes Dev. 200216: 1066-1076).

β-catenin phosphorylation by GSK3 at residues T41, S37 and S33,generates a ubiquitination degron, recruiting the E3 SCF-β-TrCP, leadingto the ubiquitination and degradation of β-catenin (Clevers H and NusseR Cell 2012, 149: 1192-1205). The inventors have further shown thatinducible ablation of CK1α in the mouse gut epithelium triggers amassive epithelial Wnt response, which surprisingly did not alterintestinal homeostasis, with only little enhanced proliferation and notumorigenesis (Elyada et al Nature 2011, 470: 409-413). This isdissimilar to the consequences of acute ablation of other components ofthe β-catenin destruction complex, such as APC, which results in loss ofhomeostasis and tumorigenesis (O. J. Sansom, O. J. et al. Genes Dev.2004, 18:1385-1390).

The inventors of the present application have found that the reason forhomeostasis maintenance following CK1α ablation is that parallel to Wntactivation, CK1α ablation induces several tumor-suppressor pathways,among which are DNA damage response (DDR), cellular senescence and p53pathway activation (Elyada E et al. Nature 2011, 470: 409-413, PribludaA et al. Cancer Cell 2013, 24: 1-5).

Whereas the molecular mechanisms underlying the activation of theseanti-neoplastic pathways are still elusive, the inventors have foundthat that CK1α ablation induces disproportionally minor DNA damage, withno signs of ATM activation, indicating that CK1α-induced DDR and p53activation likely entail uncommon molecular mechanisms (Burstain I etal, unpublished). In addition, the inventors have found that CK1αablation results in the induction of a new type of an inflammatoryresponse, denoted parainflammation, which is confined to the epithelium,with no common signs of inflammatory response (inflammatory cellinfiltration, calor, rubor, tumor and dolor) (Pribluda A et al. CancerCell 2013, 24:1-5, Lasry A and Ben-Neriah Y 2015, Trends in Immunology,Vol. 36: 217-228). Parainflammation cooperates with WT p53 activation insuppressing tumorigenesis, yet switches to a tumor promoting mechanismin the absence of functional p53 Pribiuda A et al. Cancer Cell 2013, 24:1-5, Aran et al, Genome Biol. 2016 Jul. 8; 17(1):145).

Whereas it is already established that CK1α is a major regulator of p53,the inventors have also found that the combined ablation of CK1δ andCK1ε in the gut epithelium also results in p53 activation, which maysynergize with CK1α-induced p53 activation.

IRAK1 was identified as a therapeutic target for MDS, and certainsubsets of AML and triple negative breast cancer (Garrett W. Rhyasen etal, 2013, Cancer Cell 24, 90-104, Rhyasen G W, Bolanos L, StarczynowskiD T, 2013, Exp Hematol. 41:1005-7, Zhen Ning Wee et al, 2015, NATURECOMMUNICATIONS, 6:8746). IRAK1 mRNA is over-expressed in ˜20-30% of MDSpatients and the IRAK1 protein is dramatically over-expressed and ishyperactivated in a majority of MDS marrow sample examined. IRAK1 is aserine/threonine kinase that mediates signals elicited from Toll-likereceptor (TLR) and Interleukin-1 Receptor (IL1R). Following receptoractivation, IRAK1 becomes phosphorylated which then leads to recruitmentof TRAF6, resulting in TRAF6 activation of NF-κB and JNK pathways. Themolecular source of IRAK1 overexpression and/or hyperactivation in MDS(or AML) is not conclusive. It is thought that over-expression of TLR ornecessary cofactors in MDS clones may result in chronic IRAK1 activationeven in the absence of infection. Small molecule inhibitors targetingIRAK1 (IRAK1/4 Inhibitor, Amgen Inc.) have been originally developed forautoimmune and inflammatory diseases. Given that IRAK1 is hyperactivated(i.e., phosphorylated) in MDS but not normal marrow cells, Starczynowskiand colleagues showed that IRAK-Inhibitor treatment (IRAK/4, Amgen) andthe knockdown of IRAK1 resulted in dramatic impairment of MDS cellproliferation, progenitor function, and viability in vitro and in vivo.Yu and colleagues showed that IRAK overexpression confers triplenegative breast cancer cells (TNBC) growth advantage throughNF-κB-related cytokine secretion and metastatic TNBC cells exhibit gainof IRAK1 dependency, resulting in high susceptibility to genetic andpharmacologic inhibition of IRAK1. Paclitaxel treatment of TNBC cellsinduces strong IRAK1 phosphorylation, an increase in inflammatorycytokine expression, enrichment of cancer stem cells and acquiredresistance to paclitaxel treatment. Pharmacologic inhibition of IRAK1was able to reverse paclitaxel resistance by triggering massiveapoptosis. IRAK1 was also found to be a DEK transcriptional target andis essential for head and neck cancer cell survival (Adams A K et al.Oncotarget. 2015, 22; 6(41): 43395-43407) and also as potential targetin the treatment of inflammatory- and immune-related disorders (Bahia MS et al. Cell Signal. 2015 June; 27(6):1039-55).

The inventors have thus found that compounds of the invention are ableto inhibit IRAK1, an important upstream regulator of the NF-kB pathwaywhich plays an important role in hematological malignancies.

General Description

The present invention provides a compound having the general formula(I), including any stereoisomer or salt thereof:

wherein

R₁ and R₂ are each independently selected from H, straight or branchedC₁-C₈ alkyl, straight or branched C₁-C₅ alkoxy, straight or branchedC₁-C₅ acyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl each optionally substituted byat least one of halide, hydroxyl, ester, ether, C₅-C₁₅ aryl, C₃-C₇heteroaryl, and amide; or

R₁ and R₂ together with the nitrogen atom they are connected to form a4-7 membered saturated, unsaturated or aromatic ring that may optionallyinclude at least one of N, O, NH, C═N, C═O or SO₂ and can optionally besubstituted with at least one of straight or branched C₁-C₅ alkyl,C₅-C₁₅ aryl, C₃-C₇ heteroaryl, hydroxyl, halide and cyano;

R₃ and R₄ are each independently selected from H, straight or branchedC₁-C₈ alkyl optionally substituted by at least one of halide, hydroxyl,alkoxy, C₅-C₁₅ aryl, C₃-C₇ heteroaryl, ester and amide; or

R₁ or R₂ together with R₃ and the carbon and nitrogen atom they are eachconnected to form a 4-7 membered saturated, unsaturated or aromatic ringthat may optionally include at least one of N, NH, O, C═N, C═O, SO₂, andcan optionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl, hydroxyl, carbonyl, andhalide;

R₅ and R₈ are each independently selected from H, halide, straight orbranched C₁-C₈ alkyl, straight or branched C₂-C₈ alkenyl, straight orbranched C₂-C₈ alkynyl; optionally substituted by at least one halide;

R₆ is selected from straight or branched C₁-C₈ alkyl, straight orbranched C₂-C₈ alkenyl, straight or branched C₂-C₈ alkynyl, C₅-C₁₀cycloalkyl, saturated or unsaturated 4-6 membered heterocyclyl;optionally substituted by at least one of straight or branched C₁-C₈alkyl, C₃-C₇ cycloalkyl, 4-6 membered heterocyclyl, C₅-C₁₅ aryl, C₃-C₇heteroaryl, halide, hydroxyl, C₁-C₅ alkyl halide;

R₇ is selected from straight or branched C₁-C₈ alkyl, straight orbranched C₂-C₈ alkenyl, straight or branched C₂-C₈ alkynyl; substitutedby at least one C₃-C₇ cycloalkyl, 4-6 membered heterocyclyl, C₅-C₁₅aryl, C₃-C₇ heteroaryl, halide, hydroxyl, C₁-C₅ alkyl halide.

The present invention provides a compound having the general formula(I), including any stereoisomer or salt thereof wherein:

R₁ and R₂ are each independently selected from H, straight or branchedC₁-C₈ alkyl, straight or branched C₂-C₈ alkenyl, straight or branchedC₂-C₈ alkynyl, straight or branched C₁-C₅ alkoxy, straight or branchedC₁-C₅ acyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl each optionally substituted byat least one of halide, hydroxyl, ester, ether, C₅-C₁₅ aryl, C₃-C₇heteroaryl and amide; or

R₁ and R₂ together with the nitrogen atom they are connected to form a4-7 membered saturated, unsaturated or aromatic ring that may optionallyinclude at least one of N, O, NH, C═N, C═O or SO₂ and can optionally besubstituted with at least one of straight or branched C₁-C₅ alkyl,straight or branched C₂-C₈ alkenyl, straight or branched C₂-C₈ alkynyl,C₅-C₁₅ aryl, C₃-C₇ heteroaryl, hydroxyl, halide and cyano;

R₃ and R₄ are each independently selected from H, straight or branchedC₁-C₈ alkyl, straight or branched C₂-C₈ alkenyl, straight or branchedC₂-C₈ alkynyl, optionally substituted by at least one of halide,hydroxyl, alkoxy, ester, C₅-C₁₅ aryl, C₃-C₇ heteroaryl and amide; or

R₁ or R₂ together with R₃ and the carbon and nitrogen atom they areconnected to form a 4-7 membered saturated, unsaturated or aromatic ringthat may optionally include at least one of N, NH, O, C═N, C═O, SO₂, andcan optionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, straight or branched C₂-C₅ alkenyl, straight or branchedC₂-C₅ alkynyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl, hydroxyl, carbonyl, andhalide;

R₅ and R₈ are each independently selected from H, halide, straight orbranched C₁-C₈ alkyl, straight or branched C₂-C₈ alkenyl, straight orbranched C₂-C₈ alkynyl, optionally substituted by at least one halide(in some embodiments CF₃);

R₆ is selected from straight or branched C₁-C₈ alkyl, straight orbranched C₂-C₈ alkenyl, straight or branched C₂-C₈ alkynyl, C₅-C₁₀cycloalkyl, saturated or unsaturated 4-6 membered heterocyclyl;optionally substituted by at least one of straight or branched C₁-C₈alkyl, C₃-C₇ cycloalkyl, 4-6 membered heterocyclyl, C₅-C₁₅ aryl, C₃-C₇heteroaryl, halide, hydroxyl, C₁-C₅ alkyl halide;

R₇ is selected from straight or branched C₁-C₈ alkyl, straight orbranched C₂-C₈ alkenyl, straight or branched C₂-C₈ alkynyl; substitutedby at least one C₃-C₇ cycloalkyl, 4-6 membered heterocyclyl, C₅-C₁₅aryl, C₃-C₇ heteroaryl, halide, hydroxyl, C₁-C₅ alkyl halide.

In some embodiments, R₁ and R₂ are each independently selected from H,straight or branched C₁-C₈ alkyl optionally substituted by at least oneof halide, hydroxyl, ester and amide.

In some embodiments, R₁ and R₂ are each independently selected from H,straight or branched C₁-C₅ alkoxy optionally substituted by at least oneof halide, hydroxyl, ester and amide.

In some embodiments, R₁ and R₂ are each independently selected from H,C₁-C₅ acyl, optionally substituted by at least one of halide, hydroxyl,ester, ether and amide.

In other embodiments, R₁ and R₂ are each independently selected from H,C₅-C₁₅ aryl, optionally substituted by at least one of halide, hydroxyl,ester, ether and amide.

In some embodiments, at least one of R₁ and R₂ is H.

In some embodiments, R₄ is H. In some embodiments, R₃ and R₄ are H.

In some embodiments, R₅ is selected from H, Cl and straight or branchedC₁-C₄ alkyl. In some embodiments, R₅ is H. In some embodiments, R₈ isselected from H, Cl and straight or branched C₁-C₄ alkyl. In someembodiments, R₈ is H. In some further embodiment one of R₅ or R₈ is H(i.e. only one of R₅ or R₈ is H, in other words one of R₅ or R₈ isdifferent than H).

In some embodiments, R₆ is selected from straight or branched C₁-C₈alkyl, C₅-C₁₀ cycloalkyl, saturated or unsaturated 4-6 memberedheterocyclyl; and R₇ is selected from straight or branched C₁-C₈ alkyl,substituted by at least one C₃-C₇ cycloalkyl, 4-6 membered heterocyclyl,C₅-C₁₅ aryl, C₃-C₇ heteroaryl, halide, hydroxyl, C₁-C₅ alkyl halide.

In some embodiments, R₆ is selected from a straight or branched C₁-C₈alkyl, C₅-C₁₀ cycloalkyl, 4-6 membered saturated heterocyclyl.

In some embodiments, R₇ is a straight or branched C₁-C₈ alkylsubstituted by at least one of C₃-C₇ cycloalkyl and hydroxyl.

In some embodiments, R₆ is selected from straight or branched C₁-C₈alkyl, saturated or unsaturated 4-6 membered heterocyclyl, eachoptionally substituted by at least one of straight or branched C₁-C₈alkyl, C₃-C₇ cycloalkyl, halide, hydroxyl, CF₃.

In some embodiments, R₇ is a straight or branched C₁-C₈ alkylsubstituted by at least one C₃-C₇ cycloalkyl.

In some embodiments, R₁ and R₂ together with the nitrogen atom they areconnected to form a 4-7 membered saturated ring optionally including atleast one of N or O, NH, C═N, C═O or SO₂ (i.e. in addition to the N atomR₁ and R₂ are connected to) and can optionally be substituted with atleast one of straight or branched C₁-C₅ alkyl, hydroxyl, halide andcyano.

In some embodiments, R₁ and R₂ together with the nitrogen atom they areconnected to form a 4-7 membered saturated ring.

In some embodiments, R₁ and R₂ together with the nitrogen atom they areconnected to form a 4-7 membered saturated ring including at least oneof N or O (in addition to the N atom R₁ and R₂ are connected to).

In further embodiments, R₁ and R₂ together with the nitrogen atom theyare connected to form a 4-7 membered aromatic ring optionally includingat least one of N or O (in addition to the N atom R₁ and R₂ areconnected to).

In some embodiments, R₁ or R₂ together with R₃ and the carbon andnitrogen atom they are connected to form a 4-7 membered saturated ringthat optionally includes at least one of N, NH, O, C═O, SO₂, and canoptionally be substituted with at least one of straight or branchedC₁-C₅ alkyl, hydroxyl, carbonyl, and halide.

In some embodiments, R₁ or R₂ together with R₃ and the carbon andnitrogen atom they are connected to form a 4-7 membered saturated ringthat includes at least one of NH, O, or C═O.

In some embodiments, the compound of the invention is selected from thefollowing:

In some embodiments, the compound of the invention is:

In some other embodiments, the compound of the invention is:

In other embodiments, the compound of the invention is:

The term “straight or branched C₁-C₈ alkyl” should be understood toencompass a hydrocarbon saturated chain, which can be straight orbranched, comprising 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

The term “straight or branched C₂-C₈ alkenyl” or “straight or branchedC₂-C₅ alkenyl” should be understood to encompass a hydrocarbon chainhaving at least one double bond between any two carbon atoms in thechain, which can be straight or branched, comprising 2, 3, 4, 5, 6, 7,or 8 carbon atoms or 2, 3, 4, 5 carbon atoms, respectively.

The term “straight or branched C₂-C₈ alkynyl” should be understood toencompass a hydrocarbon chain having at least one triple bond betweenany two carbon atoms in the chain, which can be straight or branched,comprising 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

The term “straight or branched C₁-C₅ alkoxy” should be understood toencompass an —OR₉ moiety wherein R₉ is a straight or branched C₁-C₅alkyl.

The term “halide” should be understood to encompass any halogen radicalselected from —F, —Br, —Cl, —I.

The term “C₁-C₅ alkyl halide” should be understood to encompass anystraight or branched alkyl chain having between 1 to 5 carbon atomsbeing substituted by at least one halogen radical selected from —F, —Br,—Cl, —I, at any point one the straight or branched chain. In someembodiments alkyl halide includes one halogen; in other embodimentsalkyl halide includes two halogen atoms (the same or different); inother embodiments, alkyl halide includes three halogen atoms (the sameor different) and so on.

The term “hydroxyl” should be understood to encompass —OH.

The term “ester” should be understood to encompass any of —C(═O)OR₁₀ or—OC(═O)R₁₀ wherein R₁₀ is a straight or branched C₁-C₈ alkyl.

The term “amide” should be understood to encompass any of—C(═O)NR₁₁R₁₂′, —NR₁₁C(═O)R₁₂′ wherein R₁₁ and R₁₂′ are eachindependently H or a straight or branched C₁-C₈ alkyl.

The term “ether” should be understood to encompass any of —R₁₃OR₁₄′ or—OR₁₅′ wherein R₁₃ is selected from a straight or branched C₁-C₈alkylene and R₁₄′ and R₁₅′ are each independently selected from astraight or branched C₁-C₈ alkyl.

The term “straight or branched C₁-C₅ acyl” should be understood toencompass any —C(═O)R₁₆ wherein R₁₆ is C₁-C₅ straight or branched alkyl.

The term “C₅-C₁₅ aryl” should be understood to encompass any single orfused aromatic ring system comprising 5 to 7 carbon atoms. Examplesinclude, but are not limited to phenyl, pentalenyl, napthalenyl,anthracenyl, and any combinations thereof.

The term “C₃-C₇ heteroaryl” should be understood to encompass any singleor fused aromatic ring system comprising 5 to 7 carbon atoms and atleast one heteroatom selected from N, O and S. Examples include, but arenot limited to furanyl, benzofuranyl, isobenzofuranyl, pyrrolynyl,indolynyl, isoindolinyl, thiophenyl, banzothiophenyl,banzo[c]thiophenyl, imidazolyl, benzimidazolyl, purinyl, pyrazolyl,indazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl,thiasolyl, benzothiazolyl, pyridinyl, auinolinyl, isoquinolinyl,pyromodinyl, quinzolinyl, pyridazinyl, cinnolinyl and any combinationsthereof.

When referring to the embodiment wherein R₁ and R₂ together with thenitrogen atom they are connected to form a 4-7 membered saturated,unsaturated or aromatic ring should be understood to relate to any ringthat may be formed having 4, 5, 6, or 7 members including said nitrogenatom. Said ring can be saturated, i.e. having all sigma bonds,unsaturated, i.e. having at least one double or at least one triple bondor any combinations thereof or aromatic, i.e. a ring system that possessaromatic character, cyclically conjugated molecular ring system with astability (due to delocalization) significantly greater than that of ahypothetical localized structure (e.g. Kekulé structure).

For example, said ring can be selected from piperidinyl, pyrrolidinyl,azetidinyl and so forth.

In some embodiments said ring may optionally include (within the ringmembers) at least one of N, O, NH, C═N, C═O or SO₂. In some furtherembodiments, said ring may be optionally substituted (on the ring systemby substitution of an —H atom on said ring) with at least one ofstraight or branched C₁-C₅ alkyl, hydroxyl, halide and cyano (—CN).

When referring to the embodiments wherein R₁ or R₂ together with R₃ andthe carbon and nitrogen atom they are connected to form a 4-7 memberedsaturated, unsaturated or aromatic ring should be understood to relateto any ring that may be formed having 4, 5, 6, or 7 members includingsaid nitrogen atom. This ring forms a spiro bi-ring system with thecyclohexyl ring in the backbone of compound of formula I. Said ring canbe saturated, i.e. having all sigma bonds, or unsaturated, i.e. havingat least one double or at least one triple bond or any combinationsthereof. In some embodiments, the ring is an aromatic ring

In some embodiments, said ring optionally includes at least one of N,NH, O, C═N, C═O, SO₂ within the ring formation. In some furtherembodiments, said ring is optionally substituted (on the ring system bysubstitution of an —H atom on said ring) with at least one of straightor branched C₁-C₅ alkyl, hydroxyl, carbonyl (—C(═O)R wherein R is H orC₁-C₅ straight or branched alkyl), and halide.

The term “C₅-C₁₀ cycloalkyl” or the term “C₃-C₇ cycloalkyl” should beunderstood to encompass a saturated (i.e. the ring containing only sigmabonds between its members) hydrocarbon ring that comprises 5, 6, 7, 8,9, or 10 carbon atoms or 3, 4, 5, 6, or 7 carbon atoms respectively.

The term “saturated, unsaturated or aromatic 4-6 membered heterocyclyl”should be understood to encompass a saturated (i.e. the ring containingonly sigma bonds between its members), unsaturated or aromatic (i.e. thering containing at least one double bond or at least one triple bond orany combinations thereof) ring containing 4, 5, or 6 members at leastone of which is a heteroatom selected from N, O, S, P.

The term “optionally substituted” as used herein means that the groupsin question are either unsubstituted or substituted with one or more ofthe substituents specified. When the groups in question are substitutedwith more than one substituent the substituents may be the same ordifferent.

Certain of the compounds described herein may contain one or more chiralcenter, or may otherwise be capable of existing as two enantiomers orseveral diastereomers. Accordingly, the compounds of this inventioninclude also mixtures of enantiomers as well as purified enantiomers orenantiomerically enriched mixtures. The compounds of this inventioninclude also mixtures of diastereomers, as well as purifieddiastereomers or diastereomerically enriched mixtures.

The invention also includes any salt of a compound of formula (I),including any pharmaceutically acceptable salt, wherein a compound ofthe invention has a net charge (either positive or negative) and atleast one counter ion (having a counter negative or positive charge) isadded thereto to form said salt. The phrase “pharmaceutically acceptablesalt(s)”, as used herein, means those salts of compounds of theinvention that are safe and effective for pharmaceutical use in mammalsand that possess the desired biological activity. Pharmaceuticallyacceptable salts include salts of acidic or basic groups present incompounds of the invention. Pharmaceutically acceptable acid additionsalts include, but are not limited to, hydrochloride, hydrobromide,hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, tartrate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucaronate, saccharate, formate, benzoate,glutamate, methanesulfonate, ethanesulfonate, benzensulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds ofthe invention can form pharmaceutically acceptable salts with variousamino acids. Suitable base salts include, but are not limited to,aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, anddiethanolamine salts. For a review on pharmaceutically acceptable saltssee BERGE ET AL., 66 J. PHARM. SCI. 1-19 (1977), incorporated herein byreference.

The invention further provides a composition comprising at least onecompound as defined herein above and below (according to general formulaI).

The present invention also relates to pharmaceutical compositionscomprising a compound of the subject invention in admixture withpharmaceutically acceptable auxiliaries, and optionally othertherapeutic agents. The auxiliaries must be “acceptable” in the sense ofbeing compatible with the other ingredients of the composition and notdeleterious to the recipients thereof.

Pharmaceutical compositions include those suitable for oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration or administration via an implant. Thecompositions may be prepared by any method well known in the art ofpharmacy.

Such methods include the step of bringing in association compounds ofthe invention or combinations thereof with any auxiliary agent. Theauxiliary agent(s), also named accessory ingredient(s), include thoseconventional in the art, such as carriers, fillers, binders, diluents,disintegrants, lubricants, colorants, flavouring agents, anti-oxidants,and wetting agents.

Pharmaceutical compositions suitable for oral administration may bepresented as discrete dosage units such as pills, tablets, dragées orcapsules, or as a powder or granules, or as a solution or suspension.The active ingredient may also be presented as a bolus or paste. Thecompositions can further be processed into a suppository or enema forrectal administration.

The invention further includes a pharmaceutical composition, ashereinbefore described, in combination with packaging material,including instructions for the use of the composition for a use ashereinbefore described.

For parenteral administration, suitable compositions include aqueous andnon-aqueous sterile injection. The compositions may be presented inunit-dose or multi-dose containers, for example sealed vials andampoules, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of sterile liquid carrier, for examplewater, prior to use. For transdermal administration, e.g. gels, patchesor sprays can be contemplated. Compositions or formulations suitable forpulmonary administration e.g. by nasal inhalation include fine dusts ormists which may be generated by means of metered dose pressurizedaerosols, nebulisers or insufflators.

The exact dose and regimen of administration of the composition willnecessarily be dependent upon the therapeutic or nutritional effect tobe achieved and may vary with the particular formula, the route ofadministration, and the age and condition of the individual subject towhom the composition is to be administered.

The invention further provides a compound as defined herein above andbelow (according to general formula I), for use in therapy.

The term “treatment” or “therapy” as used herein means the managementand care of a patient for the purpose of combating a disease, disorderor condition. The term is intended to include the delaying of theprogression of the disease, disorder or condition, the alleviation orrelief of symptoms and complications, and/or the cure or elimination ofthe disease, disorder or condition. The patient to be treated ispreferably a mammal, in particular a human being.

It should be understood that the dosage ranges set forth above areexemplary only and are not intended to limit the scope of thisinvention. The therapeutically effective amount for each active compoundcan vary with factors including but not limited to the activity of thecompound used, stability of the active compound in the patient's body,the severity of the conditions to be alleviated, the total weight of thepatient treated, the route of administration, the ease of absorption,distribution, and excretion of the active compound by the body, the ageand sensitivity of the patient to be treated, and the like, as will beapparent to a skilled artisan. The amount of administration can beadjusted as the various factors change over time.

For oral delivery, the active compounds can be incorporated into aformulation that includes pharmaceutically acceptable carriers such asbinders (e.g., gelatin, cellulose, gum tragacanth), excipients (e.g.,starch, lactose), lubricants (e.g., magnesium stearate, silicondioxide), disintegrating agents (e.g., alginate, Primogel, and cornstarch), and sweetening or flavoring agents (e.g., glucose, sucrose,saccharin, methyl salicylate, and peppermint). The formulation can beorally delivered in the form of enclosed gelatin capsules or compressedtablets. Capsules and tablets can be prepared in any conventionaltechniques. The capsules and tablets can also be coated with variouscoatings known in the art to modify the flavors, tastes, colors, andshapes of the capsules and tablets. In addition, liquid carriers such asfatty oil can also be included in capsules.

Suitable oral formulations can also be in the form of suspension, syrup,chewing gum, wafer, elixir, and the like. If desired, conventionalagents for modifying flavors, tastes, colors, and shapes of the specialforms can also be included. In addition, for convenient administrationby enteral feeding tube in patients unable to swallow, the activecompounds can be dissolved in an acceptable lipophilic vegetable oilvehicle such as olive oil, corn oil and safflower oil.

The active compounds can also be administered parenterally in the formof solution or suspension, or in lyophilized form capable of conversioninto a solution or suspension form before use. In such formulations,diluents or pharmaceutically acceptable carriers such as sterile waterand physiological saline buffer can be used. Other conventionalsolvents, pH buffers, stabilizers, anti-bacteria agents, surfactants,and antioxidants can all be included. For example, useful componentsinclude sodium chloride, acetates, citrates or phosphates buffers,glycerin, dextrose, fixed oils, methyl parabens, polyethylene glycol,propylene glycol, sodium bisulfate, benzyl alcohol, ascorbic acid, andthe like. The parenteral formulations can be stored in any conventionalcontainers such as vials and ampoules.

Routes of topical administration include nasal, bucal, mucosal, rectal,or vaginal applications. For topical administration, the activecompounds can be formulated into lotions, creams, ointments, gels,powders, pastes, sprays, suspensions, drops and aerosols. Thus, one ormore thickening agents, humectants, and stabilizing agents can beincluded in the formulations. Examples of such agents include, but arenot limited to, polyethylene glycol, sorbitol, xanthan gum, petrolatum,beeswax, or mineral oil, lanolin, squalene, and the like. A special formof topical administration is delivery by a transdermal patch. Methodsfor preparing transdermal patches are disclosed, e.g., in Brown, et al.(1988) Ann. Rev. Med. 39:221-229 which is incorporated herein byreference.

Subcutaneous implantation for sustained release of the active compoundsmay also be a suitable route of administration. This entails surgicalprocedures for implanting an active compound in any suitable formulationinto a subcutaneous space, e.g., beneath the anterior abdominal wall.See, e.g., Wilson et al. (1984) J. Clin. Psych. 45:242-247. Hydrogelscan be used as a carrier for the sustained release of the activecompounds. Hydrogels are generally known in the art. They are typicallymade by crosslinking high molecular weight biocompatible polymers into anetwork, which swells in water to form a gel like material. In someinstances, hydrogels are biodegradable or biosorbable. For purposes ofthis invention, hydrogels made of polyethylene glycols, collagen, orpoly(glycolic-co-L-lactic acid) may be useful. See, e.g., Phillips etal. (1984) J. Pharmaceut. Sci., 73: 1718-1720.

The invention further provides a compound as defined herein above andbelow (according to general formula I), for use in the inhibition of andleast one of Casein kinase I (CKI) and Interleukin-1 receptor-associatedkinase 1 (IRAK1). In some embodiments, a compound as defined hereinabove and below (according to general formula I), is used in theinhibition of CKI and IRAK1. Under the above embodiments, the use of acompound of the invention as defined herein above and below (accordingto formula I) enables the treatment of diseases, disorders, symptoms andconditions associated with at least one of CKI and IRAK1 (or in someembodiments, both CKI and IRAK1). Under such aspects the inventionprovides treatment of diseases, disorders, symptoms and conditionsassociated with the inhibition of at least one of CKI and IRAK1 (or insome embodiments, both CKI and IRAK1).

In another one of its aspects the invention provides a compound asdefined herein above and below (according to general formula I), for usein the inhibition of Interleukin-1 receptor-associated kinase 1 (IRAK1).

The invention further provides a compound as defined herein above andbelow (according to general formula I), for use in the inhibition ofCasein kinase I (CKI).

The term “Casein kinase I” should be understood to encompass a proteinkinases family that are serine/threonine-selective enzymes that functionas regulators of signal transduction pathways in most eukaryotic celltypes. CK1 isoforms are involved in Wnt signaling, circadian rhythms,nucleo-cytoplasmic shuttling of transcription factors, DNA repair, p53activation and DNA transcription.

The term “Interleukin-1 receptor-associated kinase 1” should beunderstood to encompass an enzyme encoded by the IRAK1 gene which wasfound to be an important upstream regulator of the NF-kB pathwayinvolved in disease pathways of hematological malignancies, such asmultiple myeloma, MDS, leukemia and lymphoma, breast cancer, head andneck cancer, inflammatory and immune related disorders and others.

When referring to the “inhibition” of said enzyme, it should beunderstood to encompass any qualitative or quantitative decrease in theactivity of said enzyme due to direct or indirect binding of at leastone compound of the invention to said enzyme.

The invention further provides a compound as defined herein above andbelow (according to general formula I), for use in the treatment of acondition, symptom or disease associated with a malignant condition.

In some embodiments, said malignant condition is cancer. In otherembodiments, said cancer has either WT, or mutant p53 (mutations thatdeactivate p53 typical of cancer conditions). In further embodiments,said cancer is selected from leukemia, malignant melanoma, breastcancer, prostate cancer and colorectal cancer. In some embodiments, saidcancer has WT p53.

The invention further provides a compound as defined herein above andbelow, for use in the treatment of cancer having WT p53, wherein said WTp53 is a biomarker for the said compound efficacy. Thus, under thisaspect WT p53 serves as a measurable indicator to the efficacy of cancertreatment of the compound or composition comprising a compound of theinvention. The invention further provides a method of treating cancerhaving WT p53 in a subject in need thereof, wherein said WT p53 is abiomarker for the said compound efficacy

In some embodiments, said use further comprises an induction of cancerimmunotherapy response. Thus, in some embodiments of the invention acompound or a composition comprising a compound of the inventionprovides both treatment of cancer (anti-tumor, anti-malignant activity)and cancer immunotherapy response.

In some embodiments, said malignant condition is selected fromhematological malignancies (Multiple Myeloma, Myelodysplastic Syndrome(MDS), Acute Myeloid Leukemia (AML), Melanoma and ER-negative breastcancer, Diffuse Large B cell lymphoma (DLBCL), Chronic MyelogenousLeukemia (CML), Chronic lymphocytic leukemia (CLL), head and neckcancer, and any combinations thereof.

In another one of its aspects the invention provides a compound asdefined herein above and below for use in inducing anti-tumor response.In some embodiments, said anti-tumor response comprises cancerimmunotherapy response.

The term “induced anti-tumor response” should be understood to encompassany qualitative or quantitative chemotherapy of cancerous tumors.

The term “cancer immunotherapy response” should be understood toencompass any qualitative or quantitative cancer immunotherapy inductionof the subject's own immune system to fight the cancerous cells.Typically, immunotherapies can be categorized as active, passive orhybrid (active and passive), and are designed to exploit the fact thatcancer cells often have molecules on their surface that can be detectedby the immune system of a subject, known as tumour-associated antigens(TAAs); they are often proteins or other macromolecules (e.g.carbohydrates). Active immunotherapy directs the immune system to attacktumor cells by targeting TAAs. Passive immunotherapies enhance existinganti-tumor responses.

In some embodiments, said cancer immunotherapy response relates to thechange in the expression of an immune checkpoint molecules on tumorcell, on an antigen presenting cell, on a T cell or on a Natural Killer(NK) cell.

In some embodiments, said cancer immunotherapy response relates to thereduction in the expression of an immune checkpoint molecule on tumorcell that induces suppression of the anti-tumor activity of a T cell.

In some embodiments, said cancer immunotherapy response relates to thereduction in the expression of the checkpoint protein PD-L. In someother embodiments, said immunotherapy response relates to the inhibitionof PD-L1. In some further aspects, a compound of the invention is usedin a method of inhibiting PD-L1.

The invention further provides a compound as defined herein above andbelow (according to general formula I), for use in the treatment of aninflammatory and immune related disorder including a condition, symptomor disease associated therewith.

When referring to “inflammatory and immune related disorders” it shouldbe understood to relate to any type of disorder (including conditions,symptoms and diseases associated therewith) that are treatable withInterleukin-1 receptor associated kinase inhibitors. It has been shownfor example that IRAK1 is an indispensable element of IL-Rs and TLRpathways that can regulate the abnormal levels of cytokines, andtherefore can be employed to manage immune- and inflammation-relateddisorders such as for example rheumatoid arthritis, inflammatory boweldisease, psoriasis, gout, asthma and cancer (Bahia M S et al. CellSignal. 2015 June; 27(6):1039-55).

The invention further provides a method of treating a condition, symptomor disease associated with a malignant condition in a subject in needthereof, said method comprising the step of administering to saidsubject at least one compound as defined herein above and below(according to general formula I).

The invention further provides a method of inhibiting at least one ofCasein kinase I (CKI) and Interleukin-1 receptor-associated kinase 1(IRAK1) in a subject in need thereof comprising the step ofadministrating to said subject at least one compound as defined hereinabove and below (according to general formula I).

In another one of its aspects the invention provides a method ofinhibiting Interleukin-1 receptor-associated kinase 1 (IRAK1) in asubject in need thereof comprising the step of administrating to saidsubject at least one compound as defined herein above and below(according to general formula I).

In some embodiments a method of the invention further comprisinginducing cancer immunotherapy response in said subject.

In another one of its aspects the invention provides a method forinducing an cancer immunotherapy response in a subject in need thereof,said method comprising the step of administering to said subject atleast one compound as disclosed herein above and below.

The invention further provides a method of treating an inflammatory andimmune related disorder including a condition, symptom or diseaseassociated therewith; said method comprises administering to a subjectin need thereof a compound as defined herein above and below (accordingto general formula I).

The invention also provides a method of inhibiting Casein kinase I in asubject in need thereof comprising the step of administrating to saidsubject at least one compound as defined herein above and below(according to general formula I).

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosedherein and to exemplify how it may be carried out in practice,embodiments will now be described, by way of non-limiting example only,with reference to the accompanying drawings, in which:

FIGS. 1A and 1B show a dose response of the indicated compounds of theinvention (FIG. 1A compounds A36, A39, A6, A14, A35, A51, A29, A19-4,A41, A28, A42, A43, A46; FIG. 1B compounds A30-1, A30-2, A51, A60, A64,A65)—screen in RKO colorectal cell line. RKO cells were incubated for 16hours at 37° C. with indicated concentrations of the compounds andanalyzed by Western Blot. Shown are β-catenin and p53 stabilization andphosphorylation of H2AX (γH2AX), a marker of DNA damage, indicative ofCKIα kinase inhibition. Note that whereas β-catenin and p53stabilization, and H2AX phosphorylation is a common effect of mostcompounds, some compounds do not stabilize β-catenin, whereas closeanalogues (e.g., A19-4), only stabilize β-catenin. CKIα protein levelsserves as a loading control.

FIGS. 2A-2D show that the CKIα inhibitor A14 induces apoptosis of bonemarrow cells isolated from CML blast crisis mice in a dose dependentmanner (ex vivo). FIG. 2A is a schematic representation of theexperimental procedure; Incubation with A14 or DMSO for 8 hours. FIG. 2Bshows a dramatic reduction of the leukemic cell number following A14treatment—a dose response curve. FIG. 2C shows the increased percentageof apoptotic cells (Annexin5+/7AAD−) in a dose dependent manner. FIG. 2Dshows the increased percentage of total death (7AAD+) cells in a dosedependent manner.

FIGS. 3A-3C demonstrate that A14 significantly reduces the leukemia cellburden in the peripheral blood and bone marrow in vivo in CML blastcrisis mice. FIG. 3A is a schematic representation of the experimentalprocedure, inoculating BM cells from a CML blast crisis mouse (GFP+cells) to normal C57Bl/6 mice and daily treatment (oral administration)with A14 or vehicle alone. FIG. 3B shows the percentage of GFP+ leukemiacells in peripheral blood at day 9 following treatment; A14 (N=6)compared to the vehicle treated mice (N=6). FIG. 3C shows the percentageof GFP+ leukemia cells in the bone marrow at day 9 following treatment:A14 (N=6) compared to the vehicle treated mice (N=6).

FIGS. 4A-4F show the complete blood count at day 9 following A14treatment of CML blast crisis mice: FIG. 4A shows the absolute number ofWhite blood cells (WBC) in peripheral blood (10⁹/L) (N=5). FIG. 4B showsthe absolute number of Lymphocytes (Lymph) in peripheral blood (10⁹/L)(N=5). FIG. 4C shows the absolute number of Monocytes (Mono) inperipheral blood (10⁹/L) (N=5). FIG. 4D shows the absolute number ofGranulocytes (Gran) in peripheral blood (10⁹/L). FIG. 4E shows the redblood counts (RBC, 10¹²/L). FIG. 4F shows the hemoglobin level (HGB,g/L).

FIG. 5 shows representative photographs of blood smears from A14 treatedcompared to vehicle-treated CML blast crisis mouse at day 9 following BMtransplantation.

FIG. 6 shows a schematic representation of preparation of the AML mousemodel.

FIGS. 7A-7D show the inhibitory effect of A14 on the progression of AML.FIG. 7A is a schematic representation of the experimental procedures.FIG. 7B demonstrates the percentage of GFP⁺ leukocytes in peripheralblood (PB) of A14 treated compared to vehicle treated mice. FIGS. 7C and7D shows representative Flow Cytometry analysis plots of the GFP⁺leukocytes in PB one day before (FIG. 7C) and three days (FIG. 7D)following A14 treatment.

FIGS. 8A-8G show the complete blood count at day 9 after the treatmentof AML mice with A14 of the invention. FIG. 8A shows the absolute numberof white blood cells (WBC) in peripheral blood (10⁹/L). FIG. 8B showsthe absolute number of lymphocytes (Lymph) in peripheral blood (10⁹/L).FIG. 8C shows the absolute number of monocytes (Mono) in peripheralblood (10⁹/L). FIG. 8D shows the absolute number of granulocytes (Gran)in peripheral blood (10⁹/L). FIG. 8E shows the red blood counts (RBC,10¹²/L). FIG. 8F shows the hemoglobin (g/L). FIG. 8G shows platelets(PLT) in peripheral blood (10⁹/L).

FIG. 9 shows representative photographs of blood smears from A14compared to vehicle-treated AML mice at day 9 following the first A14treatment.

FIG. 10 shows IRAK1 inhibition by the inhibitor compounds of theinvention, A51 and A14. Cells were harvested and analyzed by Westernblot. Blots were incubated with the following antibodies: Phospho-IRAK1(Thr209), (A 1074, AssayBiothechnology; 1/1,000), Phospho-IKKα/β(Ser176/180) (16A6, Cell Signaling; 1/1,000), IKKα (2682, cellsignaling; 1/1, 1000), IKK β (2370, cell signaling; 1/1, 1000),Phospho-c-Jun (Ser 63) (9261, cell signaling; 1/1, 1000), p53 (DO-1&1801hybridoma mix; dilution of 1:20 of supernatants from each), CKIα (C-19;1/1,000; Santa Cruz Biotechnology) and phospho-histoneH2AX (S139;1/1,000; Millipore). Secondary antibodies were HRP-linked goatanti-mouse, goat anti-rabbit and rabbit anti-goat antibodies (all1/10,000; Jackson). Inhibition of Phospho-IRAK1, Phospho-IKKα/β andPhospho-c-Jun indicate IRAK1 inhibition. p53 stabilization andphosphorylation of H2AX (γH2AX), a marker of DNA damage, indicative ofCKIα kinase inhibition. CKIα protein levels serves as a loading control.

FIGS. 11A-11E relate to the experimental results achieved from asingle-dose treatment of CKI inhibitor A51 in AML mice. FIG. 11Aschematically shows the preparation of AML mice and their treatment withA51 (20 mg/Kg) at day 30 from induction of the disease (Leukemiainoculation). FIGS. 11B-11E show the effect of A51 after 16 hrs fromtreatment (in comparison with treatment with vehicle alone) in reductionof the total leukemia cells in the blood: FIG. 11B shows the reductionin WBC in the peripheral blood (PB), FIG. 11C shows the shrinking of theleukemic spleen and FIG. 11D and 11E show the reduction of proportion ofleukemic blasts (GFP+ cells) in the peripheral blood (PB) and bonemarrow (BM), respectively.

FIGS. 12A and 12B show pictures of spleen and bone from treated AMLmice. Actual reduction in spleen size (splenomegaly) after treatmentwith A51, as disclosed above (FIG. 12A) and opaque bones turned tonormal color following a single dose treatment (FIG. 12B).

FIGS. 13A-13E show the results of in vitro treatment effects of CKIinhibitors on AML cells isolated from the bone marrow of leukemic mice.Shown are the percentage of dead cells (7AAD+) and the effects of theinhibitors on the expression of the major immune checkpoint proteinPD-L1 in the leukemic cells; Inhibitor treatment was in 10 or 100 nM forseveral time points (5 hours, —FIG. 13A, 6 hours, —FIGS. 13B and 13C,and 9 hours FIGS. 13D and 13E).

DETAILED DESCRIPTION OF EMBODIMENTS Preparation of(E)-1-cyclopropyl-4-(dimethylamino)-3-(2-(methylthio)pyrimidin-4-yl)but-3-en-2-one(Core A) and4-(5-(cyclopropylmethyl)-1H-pyrazol-4-yl)-2-(methylthio)pyrimidine (CoreB)

Step 1: N, O-Dimethylhydroxylamine hydrochloride (25.14 g, 257.69 mmol,1.72 eq), HATU (56.97 g, 149.82 mmol, 1.00 eq) and TEA (45.48 g, 449.46mmol, 3.00 eq) were added to a solution of 2-cyclopropylacetic acid(15.00 g, 149.82 mmol, 1.00 eq) in DCM (500 mL) at 0° C., and then themixture was stirred at 30° C. for 3 h. The resulting mixture was pouredinto water (500 mL). The aqueous washing phase was extracted with DCM(3*250 mL). The combined organic layers were dried over anhydrousNa₂SO₄, filtered and concentrated. The residue was purified by columnchromatography (SiO₂, petroleum ether (PE):ethyl acetate (EA)=50:1 to10:1) to give the desired compound 2 (13.20 g, 82.97 mmol, 55.4% yield)as colorless liquid.

¹H NMR (CDCl₃, 400 MHz) δ 3.65 (s, 3H), 3.17 (s, 3H), 2.33 (d, J=6.8 Hz,2H), 1.09-1.06 (m, 1H), 0.54-0.50 (m, 2H), 0.16-0.14 (m, 2H).

Step 2: To a solution of 4-methyl-2-methylsulfanyl-pyrimidine (9.00 g,64.19 mmol, 1.00 eq) in THF (500 mL) was added LDA (2 M, 48.46 mL, 1.51eq) at −78° C. After stirring for 1 h, a solution of compound 2 (13.79g, 96.29 mmol, 1.50 eq) in THF (500 mL) was added drop wise at −78° C.and then the reaction mixture was stirred at −78° C. for 4 h. Quenchedwith saturated aq. NH₄Cl (100 mL), the aqueous phase was extracted withethyl acetate (3×50 mL). The combined organic layers were washed withbrine, dried over anhydrous Na₂SO₄, filtered and concentrated. Theresidue was crystallized from petroleum ether/ethyl acetate to affordthe desired compound 4 (13.60 g, 55.06 mmol, 85.8% yield) as a yellowsolid.

LCMS: RT=0.629 min, m/z 223.0 [M+H]⁺.

Step 3: A solution of compound 4 (13.60 g, 61.18 mmol, 1.00 eq) inDMF-DMA (51.42 g, 2.45 mol, 40 eq) was stirred at 90° C. for 2 h. Thesolvent was removed in vacuum. The residue was purified by columnchromatography on silica gel to give Core A (10.60 g, 36.30 mmol, 59.3%yield) as a yellow solid.

LCMS: RT=0.634 min, m/z 278.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.38 (d, J=6.8 Hz, 1H), 7.62 (s, 1H), 6.96 (s,1H), 2.96-2.87 (m, 6H), 2.56 (s, 3H), 2.38 (d, J=8.8 Hz, 2H), 1.04-1.02(m, 1H), 0.52-0.46 (m, 2H), 0.09-0.04 (m, 2H).

Step 4: A solution of Core A (10.60 g, 38.21 mmol, 1.00 eq) andhydrazine hydrate (6.75 g, 114.63 mmol, 3.00 eq) in ethanol (200 mL) wasstirred at 90° C. for 3 h. The solvent was removed in vacuum. Theresidue was purified by column chromatography on silica gel to affordCore B (9.00 g, 35.81 mmol, 93.7% yield) as a light yellow solid.

LCMS: RT=2.018 min, m/z 247.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.43 (d, J=5.2 Hz, 1H), 8.04 (s, 1H), 7.10 (d,J=5.2 Hz, 1H), 3.12 (d, J=7.2 Hz, 2H), 2.60 (s, 3H), 1.19-1.14 (m, 1H),0.66 (q, J=5.2 Hz, 2H), 0.32 (q, J=5.2 Hz, 2H).

Preparation of4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-v)-2-(methylsulfonyl)pyrimidine(Core C)

Step 1: A solution of Core A (6.20 g, 22.35 mmol, 1.00 eq) andmethylhydrazine (8.00 g, 69.46 mmol, 3.11 eq) in ethanol (100 mL) wasstirred at 90° C. for 16 hrs. The solvent was removed in vacuum. Theresidue was purified by pre-HPLC (basic condition) to afford compound 5(1.80 g, 6.84 mmol, 30.6% yield) as a yellow solid and the isomer 5A(2.00 g, 7.30 mmol, 32.6% yield) as a yellow oil.

Compound 5:

LCMS: RT=2.551 min, m/z 261.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.38 (d, J=5.2 Hz, 1H), 7.90 (s, 1H), 7.11 (d,J=5.2 Hz, 1H), 3.93 (s, 3H), 3.24 (d, J=6.4 Hz, 2H), 2.62 (s, 3H),1.12-1.09 (m, 1H), 0.54-0.49 (m, 2H), 0.32-0.28 (m, 2H).

Regioisomer 5A:

LCMS: RT=2.486 min, m/z 261.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.41 (d, J=5.2 Hz, 1H), 7.90 (s, 1H), 7.04 (d,J=5.2 Hz, 1H), 3.92 (s, 3H), 2.96 (d, J=6.4 Hz, 2H), 2.61 (s, 3H),1.20-1.17 (m, 1H), 0.50-0.45 (m, 2H), 0.26-0.22 (m, 2H).

Step 2: To a solution of compound 1 (1.50 g, 5.76 mmol, 1.00 eq) in DCM(20 mL) was added m-CPBA (2.98 g, 17.28 mmol, 3.00 eq) at 0° C. andstirred at 30° C. for 2 hrs. The resulting mixture was washed withNaHSO₃ (2×100 mL), saturate aq. NaHCO₃ (100 mL) and brine, dried overanhydrous Na₂SO₄, filtered and concentrated. The residue was purified bycolumn chromatography on silica gel (petroleum ether:ethyl acetate=10:1to 1:1) to give the compound Core C (1.50 g, 5.08 mmol, 88.2% yield) asa yellow solid.

LCMS: RT=1.891 min, m/z 293.0 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.74 (d, J=5.6 Hz, 1H), 7.97 (s, 1H), 7.58 (d,J=5.6 Hz, 1H), 3.94 (s, 3H), 3.37 (s, 3H), 3.25 (d, J=6.8 Hz, 2H),1.14-1.11 (m, 1H), 0.52-0.49 (m, 2H), 0.37-0.35 (m, 2H).

General Procedures for the Preparation of A-n:

Method A: Preparation of Compound n_6

A solution of Core B (25 mmol, 1.00 eq) in DMF (50 mL) was cooled to 0°C., and NaH (1.50 eq) was added. After stirred at 0° C. for 1 h, RBr(1.60 eq) was added and the reaction mixture was stirred at 20° C. for15 hrs. Water (10.00 mL) was added and extracted with ethyl acetate(3*10 mL). The combined organic layers were washed with water (10 mL)and brine (10 mL), dried over Na₂SO₄, filtered and concentrated to givethe crude product, which was purified to give compound n_6.

Method B: Preparation of Compound n_7

To a solution of compound n_6 (10 mmol, 1.00 eq) in dichloromethane (30mL) was added m-CPBA (3.00 eq) at 0° C. and the reaction mixture wasstirred at 20-30° C. for 5 h. The resulting mixture was washed withNaHSO₃ (2×50.00 mL), saturate aq. NaHCO₃ (50 mL) and brine (10 mL),dried over anhydrous Na₂SO₄, filtered and concentrated to give compoundn_7.

Method C: Preparation of Compound A-n

A mixture of compound n_7 (10 mmol, 1.0 eq), DIEA (10.00 eq) andtrans-4-amino-cyclohexanol (1.0-3.0 eq) in DMSO (80 mL) was heated at160° C. for 3 hrs. The reaction mixture was cooled to ambienttemperature and poured into water (100 mL) and then extracted withdichloromethane (3×50 ml). The combined layers were washed with brine,dried over anhydrous Na₂SO₄, filtered and concentrated. The residue waspurified by pre-HPLC (basic condition) to afford compound A-n.

Method D: Preparation of Compound A-n

The solution of compound n_7 (10 mmol, 1.00 eq) andtrans-cyclohexane-1,4-diamine (2.0˜4.0 eq) in dioxane (40 mL) wasstirred at 130° C. for 2 h with microwave. The mixture was filtered andthe filtrate was purified by pre-HPLC (basic condition) to give A-n.

Preparation of (1r,4r)-N¹-(4-(5-(cyclopropylmethyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-cyclohexane-1,4-diamine(A-35)

Step 1: Compound 35_6 was synthesized according to the proceduredescribed in method A and it was obtained as a yellow solid after HPLC(TFA condition) purification. Yield: 17.1%;

LCMS: RT=1.959 min, m/z 289.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.49-8.47 (d, J=2.8 Hz, 1H), 8.06 (s, 1H),7.21-7.20 (d, J=5.6 Hz, 1H), 4.63-4.60 (t, J=6.6 Hz, 1H), 3.26-3.24 (d,J=6.4 Hz, 2H), 2.65 (s, 3H), 1.56 (s, 3H), 1.55 (s, 3H), 1.07-1.04 (m,1H), 0.54-0.51 (m, 2H), 0.30-0.28 (m, 2H).

Step 2: Compound 35_7 was synthesized according to the proceduredescribed in method B and it was obtained as a yellow solid afterpurification through TLC. Yield: 57.8%;

LCMS: RT=0.711 min, m/z 321.1 [M+H]⁺

Step 3: Compound A-35 was synthesized according to the proceduredescribed in method D with trans-cyclohexane-1,4-diamine (4.0 eq) and itwas obtained as a white solid. Yield: 19.7%

LCMS: RT=1.223 min, m/z 355.3 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.17-8.16 (d, J=5.2 Hz, 1H), 7.89 (s, 1H),6.72-6.71 (d, J=5.2 Hz, 1H), 4.85-4.83 (d, J=8.8 Hz, 1H), 4.60-4.53 (m,1H), 3.23-3.22 (d, J=6.4 Hz, 2H), 2.20-2.18 (d, J=10 Hz, 2H), 2.02-1.99(m, 2H), 1.54 (s, 3H), 1.52 (s, 3H), 1.40-1.27 (m, 4H), 1.08-1.06 (m,1H), 0.52-0.48 (m, 2H), 0.28-0.26 (m, 2H).

Preparation of (1R,4R)—N¹-(4-(1-cyclopentyl-5-(cyclopropylmethyl)-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-36)

Step 1: Compound 36_6 was synthesized according to the proceduredescribed in method A and it was obtained as a yellow oil afterpurification by prep_HPLC (TFA condition). Yield: 14.7%

LCMS: RT=2.228 min, m/z 315.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.31-8.30 (d, J=5.6 Hz, 1H), 7.84 (s, 1H),7.03-7.01 (d, J=5.2 Hz, 1H), 4.67-4.63 (m, 1H), 3.20-3.18 (d, J=6.4 Hz,2H), 2.52 (s, 3H), 2.05-2.02 (m, 7H), 1.65-1.62 (m, 2H), 0.98 (m, 1H),0.43-0.39 (m, 2H), 0.23-0.20 (m, 2H).

Step 2: Compound 36_7 was made according to the procedure described inmethod B and it was obtained as a yellow solid after purificationthrough column chromatography. Yield: 66.6%

LCMS: RT=0.707 min, m/z 347.2 [M+H]⁺

Step 3: Compound A-36 was made according to the procedure described inmethod D with trans-cyclohexane-1,4-diamine (2.0 eq) and it was obtainedas a whit solid after purification by prep_HPLC (TFA condition). Yield:41.35%

LCMS: RT=0.591 min, m/z 381.4 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.26 (s, 1H), 8.08-8.06 (d, J=6.4 Hz, 1H),7.27-7.25 (d, J=6.8 Hz, 1H), 4.15 (s, 1H), 3.33 (s, 3H), 3.20 (s, 1H),2.25-1.99 (m, 11H), 1.75 (m, 2H), 1.64-1.58 (m, 4H), 1.16 (m, 1H),0.57-0.55 (m, 2H), 0.36-0.35 (m, 1H).

Preparation of (1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-39)

Step 1: To a solution of Core B (700.00 mg, 2.84 mmol, 1.00 eq) in MeCN(14 mL) was added CS₂CO₃ (1.85 g, 5.68 mmol, 2.00 eq) at 0° C. After 30min, 4-bromotetrahydropyran (703.03 mg, 4.26 mmol, 1.50 eq) was added.The mixture was stirred at 100° C. for 16 h in a sealed tube. Thereaction mixture was filtered and the filtrate was concentrated. Thecrude product was purified by prep-HPLC (TFA) to give compound 39_6(45.0 mg, 136.18 μmol, 4.8% yield) as a yellow solid.

LCMS: RT=0.702 min, m/z 331.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.42-8.40 (d, J=5.6 Hz, 1H), 7.94 (s, 1H),7.11-7.10 (d, J=5.2 Hz, 1H), 4.37-4.33 (m, 1H), 4.19-4.15 (m, 1H),3.59-3.54 (m, 2H), 3.28-3.26 (d, J=6.4 Hz, 2H), 2.6 (s, 3H), 2.44-2.40(m, 2H), 1.87-1.84 (m, 2H), 1.05-1.02 (m, 1H), 0.55-0.50 (m, 2H),0.33-0.30 (m, 2H).

Step 2: Compound 39_7 was made according to the procedure described inmethod B and it was obtained as a yellow solid after prep TLCpurification. Yield: 72.6%

LCMS: RT=0.607 min, m/z 363.1 [M+H]⁺

Step 3: Compound A-39 was made according to the procedure described inmethod C with trans-cyclohexane-1,4-diamine (3.0 eq) without DIEA and itwas obtained as a yellow solid after purification through prep-HPLC(basic condition). Yield: 40.2%

LCMS: RT=1.104 min, m/z 397.4 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.17-8.16 (d, J=4.4 Hz, 1H), 7.89 (s, 1H),6.72-6.70 (d, J=5.2 Hz, 1H), 5.12-5.07 (m, 1H), 4.37-4.34 (m, 1H),4.18-4.15 (m, 2H), 3.84 (s, 1H), 3.59-3.53 (m, 2H), 3.26-3.25 (d, J=6.4Hz, 2H), 2.77 (s, 1H), 2.44-2.40 (m, 2H), 2.16 (s, 2H), 1.93 (s, 2H),1.87-1.83 (m, 2H), 1.32-1.27 (m, 4H), 1.05 (s, 1H), 0.54-0.49 (m, 2H),0.29-0.27 (m, 2H).

Preparation of (1R,4R)—N′-(4-(5-(cyclopropylmethyl)-1-(oxetan-3-yl)-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-43)

Step 1: Compound 43_6 was synthesized according to the proceduredescribed in method A and it was obtained as a yellow solid afterpurification by prep-HPLC (TFA condition). Yield: 17.9%

LCMS: RT=0.664 min, m/z 303.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.44-8.42 (d, J=5.2 Hz, 1H), 8.04 (s, 1H),7.13-7.12 (d, J=5.6 Hz, 1H), 5.89-5.34 (m, 1H), 5.28-5.25 (t, J=6.6 Hz,2H), 5.03-5.00 (t, J=7.0 Hz, 2H), 3.21-3.20 (d, J=6.4 Hz, 2H), 2.6 (s,3H), 0.97-0.94 (m, 1H), 0.51-0.46 (m, 2H), 0.25-0.22 (m, 2H).

Step 2: Compound 43_7 was synthesized according to the proceduredescribed in method B and it was obtained as a yellow solid after prepTLC purification. Yield: 79.6%

LCMS: RT=0.566 min, m/z 335.1 [M+H]⁺

Step 3: Compound A-43 was synthesized according to the proceduredescribed in method D with trans-cyclohexane-1,4-diamine (3.0 eq) and itwas obtained as a white gum after purification by prep-HPLC (TFAcondition). Yield: 24.7%;

LCMS: RT=0.987 min, m/z 369.3 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.40 (s, 1H), 8.13-8.11 (d, J=6.8 Hz, 1H),7.31-7.29 (d, J=7.2 Hz, 1H), 5.83-5.80 (t, J=7.0 Hz, 1H), 5.15-5.12 (t,J=6.2 Hz, 2H), 5.06-5.03 (t, J=7.0 Hz, 2H), 4.12 (s, 1H), 3.28-3.26 (d,J=6.4 Hz, 2H), 3.19 (s, 1H), 2.23-2.17 (m, 4H), 1.62-1.60 (m, 4H), 1.06(s, 1H), 0.53-0.52 (m, 2H), 0.28-0.27 (m, 2H).

Preparation of (R)-tetrahydrofuran-3-yl methanesulfonate (41_4)

To a solution of (R)-tetrahydrofuran-3-ol (500.00 mg, 5.68 mmol, 1.00eq) in DCM (5 mL) were added TEA (1.15 g, 11.36 mmol, 2.00 eq) andmethanesulfonyl chloride (650.11 mg, 5.68 mmol, 1.00 eq) at 0° C. Themixture was stirred at 20° C. for 1 hr. The mixture was diluted withwater (20 mL) and extracted with DCM (10 mL*2). The combined organiclayers were concentrated to give 41_4 (900.00 mg, 5.42 mmol, 95.3%yield) as a colorless liquid.

¹H NMR (CDCl₃, 400 MHz) δ 5.33-5.30 (m, 1H), 4.06-3.90 (m, 4H), 3.05 (s,3H), 2.28-2.23 (m, 2H).

Preparation of (1R,4S)—N¹-(4-(5-(cyclopropylmethyl)-1-((S)-tetrahydrofuran-3-yl)-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-41)

Step 1: Compound 41_6 was synthesized according to the proceduredescribed in method A and it was obtained as a yellow solid afterpurification by prep-HPLC (TFA condition). Yield: 13.1%

¹H NMR (CDCl₃, 400 MHz) δ 8.42-8.40 (d, J=4.4 Hz, 1H), 7.94 (s, 1H),7.11-7.10 (d, J=7.2 Hz, 1H), 5.04-5.00 (m, 1H), 4.27-4.03 (m, 4H),3.29-3.27 (d, J=8.4 Hz, 2H), 2.6 (s, 3H), 2.48-2.42 (m, 2H), 1.07-1.02(m, 1H), 0.53-0.49 (m, 1H), 0.30-0.28 (m, 2H).

Step 2: Compound 41_7 was synthesized according to the proceduredescribed in method B and it was obtained as a yellow solid after prepTLC purification. Yield: 57.5%

LCMS: RT=0.585 min, m/z 349.2 [M+H]⁺

Step 3: Compound A-41 was synthesized according to the proceduredescribed in method D with trans-cyclohexane-1,4-diamine (4.0 eq) and itwas obtained as a white gummy solid after purification by prep HPLC (TFAcondition). Yield: 17.2%

LCMS: RT=0.969 min, m/z 383.3 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.26 (s, 1H), 8.10-8.09 (d, J=6.8 Hz, 1H),7.25-7.23 (d, J=6.8 Hz, 1H), 5.28-5.23 (m, 1H), 4.22-4.12 (m, 3H),3.99-3.95 (m, 2H), 3.35-3.33 (d, J=6.8 Hz, 2H), 3.19 (s, 1H), 2.47-2.44(m, 2H), 2.35-2.17 (m, 4H), 1.62-1.57 (m, 4H), 1.14 (s, 1H), 0.58-0.56(m, 2H), 0.36 (m, 2H).

Preparation of (S)-tetrahydrofuran-3-yl methanesulfonate (42_4)

Compound 42_4 was made in the same way as 41_4 from(S)-tetrahydrofuran-3-ol and it was a colorless liquid. Yield: 90.0%

¹H NMR (CDCl₃, 400 MHz) δ 5.33-5.30 (m, 1H), 4.04-3.86 (m, 4H), 3.05 (s,3H), 2.27-2.22 (m, 2H).

Preparation of (1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-((R)-tetrahydrofuran-3-yl)-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-42)

Step 1: Compound 42_6 was synthesized according to the proceduredescribed in method A and it was obtained as a yellow solid afterpurification by prep HPLC (TFA condition). Yield: 21.4%

LCMS: RT=0.692 min, m/z 317.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.42-8.40 (d, J=4.4 Hz, 1H), 7.95 (s, 1H),7.12-7.10 (d, J=7.2 Hz, 1H), 5.04-5.00 (m, 1H), 4.27-4.03 (m, 4H),3.29-3.27 (d, J=8.4 Hz, 2H), 2.6 (s, 3H), 2.47-2.42 (m, 2H), 1.06-1.03(m, 1H), 0.55-0.50 (m, 1H), 0.31-0.29 (m, 2H).

Step 2: Compound 42_7 was synthesized according to the proceduredescribed in method B and it was obtained as a yellow solid after prepTLC purification. Yield: 83.2%

LCMS: RT=0.587 min, m/z 349.1 [M+H]⁺

Step 3: Compound A-42 was synthesized according to the proceduredescribed in method D and it was obtained as a white gummy solid afterpurification by prep HPLC (TFA condition). Yield: 51.5%

LCMS: RT=1.078 min, m/z 383.3 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.26 (s, 1H), 8.10-8.08 (d, J=6.8 Hz, 1H),7.28-7.26 (d, J=6.8 Hz, 1H), 5.27-5.24 (m, 1H), 4.22-4.13 (m, 3H),3.99-3.96 (m, 2H), 3.35-3.31 (t, J=6.8 Hz, 2H), 3.19 (s, 1H), 2.46-2.44(m, 2H), 2.36-2.17 (m, 4H), 1.63-1.58 (m, 4H), 1.15 (s, 1H), 0.58-0.56(m, 2H), 0.36 (m, 2H).

Preparation of (1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-(tetrahydro-2H-pyran-3-yl)-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-38)

Step 1: The mixture of 38_1 (1.20 g, 11.99 mmol, 1.40 eq) and benzylN-aminocarbamate (1.42 g, 8.56 mmol, 1.00 eq) in MeOH (15 mL) wasstirred at 30° C. for 2 h. NaBH₃CN (2.69 g, 42.82 mmol, 5.00 eq) wasadded. The resulting mixture was stirred at 30° C. for 16 h. The mixturewas concentrated and diluted with water (50 mL) and EA (50 mL). Theorganic layer was concentrated and purified by column chromatography(PE:EA=10:1˜2:1) to give 38_2 (1.50 g, 5.99 mmol, 70% yield) as acolorless oil.

LCMS: RT=0.568 min, m/z 273.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 7.40-7.35 (m, 5H), 6.25 (s, 1H), 5.26-5.14 (m,2H), 3.86-3.73 (m, 3H), 3.48-3.30 (m, 2H), 1.91-1.85 (m, 2H), 1.59-1.46(m, 2H).

Step 2: To a solution of 38_2 (1.60 g, 6.39 mmol, 1.00 eq) in MeOH (15mL) was added Pd(OH)₂ (179.54 mg, 1.28 mmol, 0.20 eq). The mixture wasstirred at 20° C. under H₂ (15 psi) for 16 h. The mixture was filteredand the filtrate was concentrated to give 38_3 (450 mg, crude) as acolorless oil, which was used in the next step directly withoutpurification.

Step 3: Compound 38_6 was made from Core A and compound 38_4 accordingto the same procedure as described for the synthesis of Core B and itwas obtained as a yellow oil after purification by prep-HPLC (TFAcondition). Yield: 40.3%

LCMS: RT=0.851 min, m/z 331.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.41-8.40 (d, J=5.2 Hz, 1H), 7.92 (s, 1H),7.10-7.09 (d, J=5.2 Hz, 1H), 4.34-4.33 (m, 1H), 4.03-4.00 (m, 2H),3.87-3.81 (t, J=10.8 Hz, 1H), 3.51-3.50 (m, 1H), 3.29-3.19 (m, 2H), 2.60(s, 3H), 2.35 (m, 1H), 2.13 (m, 1H), 1.89-1.85 (m, 2H), 1.06-1.03 (m,1H), 0.53-0.51 (m, 2H), 0.31 (m, 2H).

Step 4: Compound 38_7 was synthesized according to the proceduredescribed in method B and it was obtained as a yellow solid after prepTLC purification. Yield: 56.1%

LCMS: RT=0.739 min, m/z 363.1 [M+H]⁺

Step 5: Compound A-38 was synthesized according to the proceduredescribed in method D with trans-cyclohexane-1,4-diamine (4.0 eq) and itwas obtained as a yellow solid after purification by prep HPLC (basiccondition). Yield: 40%

LCMS: RT=1.154 min, m/z 397.3 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.18-8.17 (d, J=5.2 Hz, 1H), 7.86 (s, 1H),6.70-6.69 (d, J=5.2 Hz, 1H), 4.88-4.86 (m, 1H), 4.32-4.29 (m, 1H),4.02-4.00 (t, J=5.6 Hz, 2H), 3.86-3.81 (m, 2H), 3.51-3.49 (t, J=3.00 Hz,1H), 3.27-3.19 (m, 2H), 2.81 (s, 1H), 2.34-2.31 (m, 1H), 2.19-2.16 (m,3H), 2.00-1.97 (m, 4H), 1.38-1.27 (m, 4H), 1.06 (s, 1H), 0.52-0.50 (m,2H), 0.28 (m, 2H)

General Procedure for the Synthesis of A-n

Method E

The reaction mixture of Core C (1.00 eq) and amine n (4.00 eq) in DMSO(8 mL) was stirred at 160° C. for 3 h. The reaction mixture was cooledto rt and poured onto ice-H₂O (20 mL). The aqueous layer was extractedwith EA (50 mL*3). The combined organic layers were washed with brine(50 mL*3), dried over Na₂SO₄, filtered and concentrated. The residue waspurified by column chromatography to give A-n.

Method F

The reaction mixture of Core C (1.00 eq), TBAF (2.00 eq), K₂CO₃ (4.00eq) and amine n (4.00 eq) in DMSO (10 mL) was stirred at 160° C. for 3h. The reaction mixture was cooled to 15° C. and poured into H₂O (20mL). The aqueous layer was extracted EA (20 mL*3). The combined organiclayers were washed with brine (20 mL*3), dried over Na₂SO₄, filtered andconcentrated. The residue was purified by Pre-HPLC (HCl condition) togive A-n.

Method G

The reaction mixture of amine n (1.20 eq) and Core C (1.00 eq) indioxane (3 mL) was stirred at 130° C. under microwave for 1.5 h. Thereaction mixture was poured into H₂O (20 mL). The aqueous layer wasextracted EA (20 mL*3). The combined organic layers were washed withbrine (20 mL*3), dried over Na₂SO₄, filtered and concentrated. Theresidue was purified by Pre-HPLC (basic condition) to give A-n.

Preparation of (1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-14) and (1r,4r)-N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N4,N4-dimethylcyclohexane-1,4-diamine(A-28)

Step 1: Compound A-14 was prepared according to the procedure describedin method G and it was obtained as a yellow solid. Yield: 38.4%

LCMS: RT=2.043 min, m/z 327.2 [M+H]⁺.

¹H NMR (CDCl₃, 400 MHz) δ 8.17 (d, J=5.3 Hz, 1H), 7.83 (s, 1H), 6.70 (d,J=5.3 Hz, 1H), 4.91 (s, 1H), 3.93-3.85 (m, 4H), 3.21 (d, J=6.3 Hz, 2H),2.74 (s, 1H), 2.16 (d, J=4.0 Hz, 2H), 1.94 (d, J=6.7 Hz, 2H), 1.30-1.25(m, 4H), 1.11-1.10 (m, 1H), 0.50-0.46 (m, 1H), 0.27-0.24 (m, 1H)

Step 2: To a mixture of A-14 (18.52 mg, 122.53 μmol, 1.00 eq) in EtOH(500 μL) was added 2,3-dihydrobenzotriazol-1-ylmethanol (18.52 mg,122.53 μmol, 1.00 eq) in one portion at 0° C. The mixture was stirred at15° C. for 1 hr and NaBH₄ (9.27 mg, 245.06 μmol, 2.00 eq) was added. Theresulting mixture was stirred at 15° C. for 1 h, and poured into H₂O (10mL). The aqueous layer was extracted DCM (20 mL*3). The combined organiclayer was washed with brine, dried over NaSO₄ and concentrated. Theresidue was purified by prep HPLC (basic condition) to give A-28 (5.00mg, 14.10 μmol, 11.5% yield, 100% purity) as a white solid.

LCMS: RT=2.535 min, m/z 355.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.10 (d, J=5.2 Hz, 1H), 7.76 (s, 1H), 6.63 (d,J=5.2 Hz, 1H), 4.78 (d, J=3.6 Hz, 1H), 3.81 (s, 3H), 3.77-3.71 (m, 1H),3.13 (d, J=6.4 Hz, 2H), 2.27 (s, 6H), 2.17 (d, J=12.4 Hz, 2H), 1.93 (d,J=11.6 Hz, 2H), 1.36-1.31 (m, 2H), 1.23-1.17 (m, 2H), 1.04-1.02 (m, 1H),0.44-0.39 (m, 2H), 0.20-0.18 (m, 2H).

Preparation of 8-amino-1,3-diazaspiro[4,5]decane-2,4-dione hydrochloride(amine-19)

Step 1: A solution of NaCN (2.75 g, 56.02 mmol, 2.39 eq) in H₂O (10 mL)was added to the mixture of compound 19_1 (5.00 g, 23.44 mmol, 1.00 eq)and (NH₄)₂CO₃ (4.96 g, 51.57 mmol, 2.20 eq) in EtOH (25 mL) and H₂O (25mL). The reaction mixture was stirred at 10° C. for 16 hours and then at70° C. for another 24 hours. TLC (PE:EA=3:1) showed that reactant wasconsumed (Rf=0.55) and a major spot formed (Rf=0.25). The reactionmixture was allowed to cool down and filtered. The filter cake waswashed with H₂O (100 mL) and dried. Compound 19_2 (4.00 g, 14.12 mmol,60.2% yield) was obtained as a white solid.

¹H NMR (DMSO-d6, 400 MHz) δ 10.51 (s, 1H), 8.48 (s, 1H), 6.70-6.72 (d,J=8.0 Hz, 1H), 3.16 (s, 1H), 1.79-1.76 (m, 2H), 1.65-1.64 (d, J=4.0 Hz,2H), 1.62-1.52 (m, 2H), 1.60-1.59 (m, 1H), 1.37 (s, 11H).

Step 2: To the mixture of compound 19_2 (1.00 g, 3.53 mmol, 1.00 eq) inMeOH (10 mL) was added HCl/dioxane (4 M, 10 mL, 11.33 eq) at 0° C. andthe reaction mixture was stirred at 10° C. for 16 hour. LCMS showed theconsumption of reactant. The reaction mixture was concentrated to giveAmine-19 (700.00 mg, 3.19 mmol, 90.3% yield, HCl) as a white solid.

¹H NMR (DMSO-d6, 400 MHz) δ 8.52 (s, 1H), 8.21 (s, 3H), 3.06 (s, 1H),1.96-1.89 (m, 1H), 1.76-1.59 (m, 6H).

Preparation of8-((4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-1,3-diazaspiro[4.5]decane-2,4-dione(A19_4) and(1-amino-4-((4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-ylamino)cyclohexyl)methanol(A-19)

Step 1: Compound A19_4 was prepared according to the procedure describedin method E with amine-19 (4 eq) and DIEA (4 eq) and it was as a whitesolid after purification by prep HPLC (basic condition). Yield: 72.2%

LCMS: RT=0.573 min, m/z 397.2 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.22 (s, 1H), 8.14-8.09 (m, 1H), 7.24 (d,J=5.2 Hz, 1H), 5.19 (d, J=6.8 Hz, 1H), 4.21 (s, 1H), 3.94 (s, 3H), 3.30(d, J=6.8 Hz, 2H), 2.20 (d, J=6.0 Hz, 2H), 2.09-2.05 (t, 2H), 1.86 (d,J=14.8 Hz, 2H), 1.69-1.65 (m, 2H), 1.25-1.21 (m, 1H), 0.60 (d, J=7.6 Hz,2H), 0.39-0.35 (m, 2H),

Step 2: Compound A19_4 (95.00 mg, 240.23 μmol, 1.00 eq) was added toNaOH/H₂O (3 M, 640.60 μL, 8.00 eq) and the reaction mixture was stirredat 120° C. for 16 h. The reaction mixture was cooled to 15° C. andpoured into H₂O (20 mL). The aqueous layer was adjusted to pH=7 with 2NHCl solution. The mixture was concentrated. The crude product wastriturated with MeOH (50 mL*3). The filtrate was concentrated to give195 (300.00 mg, crude) as a white solid.

LCMS: RT=1.148 min, m/z 371.2 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.15 (d, J=5.2 Hz, 1H), 7.95-7.93 (m, 1H),6.89-6.85 (m, 1H), 4.03 (s, 1H), 3.91 (s, 3H), 3.28 (s, 2H), 2.32-2.25(m, 2H), 2.16-2.13 (d, J=9.6 Hz, 2H), 1.99-1.95 (d, J=9.6 Hz, 2H),1.57-1.54 (d, J=14 Hz, 2H), 1.37 (s, 1H), 0.51-0.48 (m, 2H), 0.31-0.27(m, 2H),

Step 3: To the mixture of 19_5 (330.00 mg, 890.81 μmol, 1.00 eq) in THF(500 μL) was added BH₃ (1M, 7.13 mL, 8.00 eq) and the reaction mixturewas stirred at 100° C. for 16 h. The reaction mixture was cooled to 15°C. and poured into MeOH (20 mL). The mixture was concentrated. The crudeproduct was purified by Pre-HPLC (basic condition) to give A-19 (23.00mg, 64.52 μmol, 7.2% yield, 100% purity) as a white solid.

LCMS: RT=1.060 min, m/z 357.2 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.12 (d, J=5.6 Hz, 1H), 7.93 (s, 1H),6.85-6.82 (m, 1H), 3.88-3.80 (m, 4H), 3.28 (s, 2H), 3.39 (s, 2H), 3.27(d, J=6.4 Hz, 2H), 2.03-1.94 (m, 3H), 1.68-1.58 (m, 6H), 1.13 (d, J=5.2Hz, 1H), 0.50-0.47 (m, 2H), 0.30-0.28 (m, 2H).

Preparation of8-((4-(5-(cyclopropylmethyl)-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-3-oxa-1-azaspiro[4.5]decan-2-one(A-26)

To the mixture of A-19 (20.00 mg, 56.11 μmol, 1.00 eq) in THF (500 μL)was added CDI (9.1 mg, 56.11 μmol, 1.00 eq) and the reaction mixture wasstirred at 25° C. for 16 h. The reaction mixture was concentrated. Thecrude product was purified by prep HPLC (basic condition) to give A-26(4.00 mg, 10.46 μmol, 18.6% yield, 100% purity) as a white solid.

LCMS: RT=2.210 min, m/z 383.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.19 (s, 1H), 7.84 (s, 1H), 6.74 (s, 1H), 5.97(s, 1H), 5.02 (s, 1H), 4.15 (s, 1H), 3.89 (s, 4H), 3.17 (s, 2H),2.16-2.13 (d, J=12.4 Hz, 2H), 1.98-1.95 (d, J=13.2 Hz, 2H), 1.72-1.68(d, J=12.8 Hz, 2H), 1.48-1.45 (d, J=10.08 Hz, 2H), 1.10 (s, 1H), 0.49(s, 2H), 0.24 (s, 2H).

Preparation of (1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N4-methylcyclohexane-1,4-diamine(A-27)

Step 1: Compound 27_2 was made according to the procedure described inmethod E with amine-27 (1.0 eq) and DIEA (10 eq) at 160° C. for 6 h andit was obtained as a yellow solid after purification by prep HPLC (TFA).Yield: 41.1%

¹H NMR (CDCl₃, 400 MHz) δ 8.18 (d, J=5.2 Hz, 1H), 7.83 (s, 1H), 6.71 (d,J=5.6 Hz, 1H), 4.87 (s, 1H), 4.41 (s, 1H), 3.89-3.82 (m, 4H), 3.49 (s,1H), 3.19 (d, J=6.3 Hz, 2H), 2.19-2.05 (m, 4H), 1.46 (s, 9H), 1.35-1.29(m, 7H), 1.26 (m, 2H), 1.23 (m, 2H), 0.50-0.46 (m, 2H), 0.26-0.24 (m,2H)

Step 2: To a solution of 272 (100.00 mg, 234.44 μmol, 1.00 eq) in THF(4.00 mL) was added LiAlH₄ (26.69 mg, 703.32 μmol, 3.00 eq) at 0° C. Themixture was stirred at 70° C. for 3 h. The mixture was cooled to 0° C.and then quenched with aq.NH4Cl (3 drops). The resulting solution wasdried over Na₂SO₄ and then concentrated. The residue was failed to bepurified by prep-HPLC. So A-27 (70 mg, crude) will be purified afterprotected with Boc group. To the solution of A-27 (crude) in THF (1 mL)was added (Boc₂)₂ (200 mg, 2 eq). After stirring for 1 hour at 20° C.,the reaction mixture was purified by prep-TLC (EA) to get 27_Boc (30 mg,29.13% yield total), which was used for next step directly.

Step 3: To a solution of 27_Boc (30.00 mg, 68.09 μmol, 1.00 eq) in DCM(100 μL) was added HCl/dioxane (4 M, 85 μL, 5.00 eq) in one portion at0° C. and the mixture was stirred at 15° C. for 5 h. The reactionmixture was concentrated to give A-27 (12.00 mg, 30.88 μmol, 45.35%yield, 97% purity, HCl) as a light yellow solid. Yield: 45.4%

LCMS: RT=2.558 min, m/z 341.2 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.26 (s, 1H), 8.14 (d, J=6.4 Hz, 1H), 7.31 (d,J=6.4 Hz, 1H), 4.19-4.12 (m, 1H), 3.95 (s, 3H), 3.71 (s, 2H), 3.17 (s,1H), 2.76 (s, 3H), 2.31-2.20 (m, 4H), 1.65 (m, 4H), 1.21 (s, 1H), 0.51(s, 2H), 0.37 (d, J=3.2 Hz, 2H).

Preparation of N-((1R,4R)-4-aminocyclohexyl)-2-methoxyacetamidehydrochloride (amine-29)

Step 1: To a mixture of 291 (658.25 mg, 3.07 mmol, 1.0 eq) and2-methoxyacetyl chloride (500 mg, 4.61 mmol, 1.5 eq) in DCM (5 mL) wasadded DIEA (310.82 mg, 3.07 mmol, 1.00 eq) in at 0° C. Then the reactionmixture was stirred at 15° C. for 2 hours. The reaction mixture wasconcentrated under vacuum. The residue was purified by columnchromatography to give 29_2 (490.00 mg, 1.71 mmol, 55.7% yield) as awhite solid.

¹H NMR (CDCl₃, 400 MHz) δ 3.80 (s, 1H), 3.74-3.67 (m, 1H), 3.40 (s, 1H),3.33 (s, 3H), 1.97-1.87 (m, 4H), 1.37 (s, 9H), 1.22-1.12 (m, 3H)

Step 2: Compound 29-2 was deprotected in a similar manner as shown instep 3 of the synthesis of A27 and Amine-29 was obtained as a whitesolid. Yield: 91.9%

1H NMR (CD₃OD, 400 MHz) 4.05 (s, 1H), 374 (s, 2H), 3.70-3.69 (m, 1H),3.36 (s, 3H), 3.28-3.27 (m, 1H), 3.09-3.07 (m, 1H), 2.07-2.04 (m, 2H),1.94-1.92 (m, 2H), 1.56-1.40 (m, 4H)

Preparation ofN-((1R,4R)-4-((4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)cyclohexyl)-2-methoxyacetamide(A29_1)

Compound A29_1 was made according to the procedure described in method Ewith amine-29 (1.0 eq) and DIEA (4 eq) and it was obtained as a lightyellow solid after purification by prep HPLC (basic condition). Yield:27.4%

LCMS: RT=2.535 min, m/z 355.2 [M+H]⁺

1H NMR (CDCl₃, 400 MHz) δ 8.20 (d, J=5.2 Hz, 1H), 7.85 (s, 1H), 6.73 (d,J=5.2 Hz, 1H), 6.40 (d, J=8.0 Hz, 1H), 4.92 (s, 1H), 3.91 (m, 7H), 3.45(m, 3H), 3.27-3.20 (m, 2H), 2.21 (s, 2H), 2.10 (s, 2H), 1.45-1.36 (m,4H), 1.13-1.12 (m, 1H), 0.53-0.48 (m, 2H), 0.28-0.26 (q, J=4.9 Hz, 2H)

Preparation of (1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N4-(2-methoxyethyl)cyclohexane-1,4-diamine(A-29)

To a mixture of LAH (40.00 mg, 1.05 mmol, 10.50 eq) in THF (4 mL) wasadded a solution of A29_1 (40.00 mg, 100.38 μmol, 1.00 eq) in THF (1 mL)at 15° C. The mixture was stirred at 70° C. for 36 hr. The reactionmixture was cooled down and poured into H₂O (0.1 mL). 1N NaOH (0.1 mL)was added and filtered. The filtrate was concentrated. The crude productwas purified by Pre-HPLC (basic condition) to give A-29 (4.00 mg, 9.88μmol, 9.8% yield, 95.0% purity) as a yellow solid.

LCMS: RT=2.535 min, m/z 355.2 [M+H]⁺

¹H NMR (CDCl₃, 300 MHz) δ 8.17 (d, J=6.8 Hz, 1H), 7.82 (s, 1H),6.76-6.69 (m, 1H), 4.88 (d, J=10.4 Hz, 1H), 3.89 (s, 4H), 3.54-3.50 (m,2H), 3.38 (s, 3H), 3.22 (d, J=8.40 Hz, 2H) 2.84-2.81 (m, 2H), 2.49 (s,1H) 2.19 (s, 2H), 2.01 (s, 1H) 1.38 (s, 4H), 1.07 (s, 1H) 0.49-0.43 (m,2H), 0.25-0.20 (m, 2H)

Preparation of (1R, 4R)-4-(piperidin-1-yl)cyclohexanamine hydrochloride(amine-47)

Step 1: A mixture of 47_1 (200.00 mg, 933.27 μmol, 1.00 eq),1,5-diiodopentane (302.32 mg, 933.27 μmol, 1.00 eq) and K₂CO₃ (515.95mg, 3.73 mmol, 4.00 eq) in MeCN (10 mL) was stirred at 90° C. for 16hours. The mixture was filtered and the filtrate was concentrated togive 47_2 (300 mg, crude) as a white solid and it was used in next stepdirectly.

Step 2: Crude 47_2 was deprotected in a similar manner as shown in step3 of the synthesis of A27 and amine-47 was obtained as a white solid.

¹H NMR (D₂O, 400 MHz) δ 3.55-3.10 (m, 6H), 2.16-2.08 (m, 4H), 1.59-1.41(m, 10H).

Preparation of4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)-N-((1R,4R)-4-(piperidin-1-yl)cyclohexyl)pyrimidin-2-amine(A-47)

Compound A-47 was prepared according to the procedure described inmethod F with amine-47 and it was obtained as a white solid afterpurification by prep HPLC (HCl condition). Yield: 49.3%

LCMS: RT=1.530 min, m/z 395.2 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.21 (s, 1H), 8.06 (d, J=6.8 Hz, 1H),7.24-7.21 (m, 1H), 4.10 (s, 1H), 3.88 (s, 3H), 3.49 (d, J=11.2 Hz, 2H),3.23 (s, 2H), 3.06-3.00 (m, 4H), 2.26 (d, J=10 Hz, 4H), 1.96-1.57 (m,10H), 1.15 (s, 1H), 0.50 (s, 2H), 0.31-0.30 (m, 2H)

Preparation of (1R,4R)-4-morpholinocyclohexanamine hydrochloride(amine-48)

Crude amine-48 was synthesized in a similar manner as described for thesynthesis of amine-47 and it was obtained as a yellow solid.

¹H NMR (D₂O, 400 MHz) δ 3.87 (s, 4H), 3.24-3.11 (m, 6H), 2.20-2.12 (m,4H), 1.59-1.39 (m, 5H).

Preparation of4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)-N-((1r,4r)-4-morpholinocyclohexyl)pyrimidin-2-amine (A-48)

Compound A-48 was prepared according to the procedure described inmethod F with amine-48 with TBAF (1.0 eq) and it was obtained as a whitesolid after purification by prep HPLC (HCl condition). Yield: 51.2%

LCMS: RT=2.095 min, m/z 397.3 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.16 (s, 1H), 8.01 (s, 1H), 7.19 (s, 1H),4.06-3.97 (m, 3H), 3.83-3.78 (m, 5H), 3.51-3.42 (m, 2H), 3.24 (s, 1H),3.14-3.12 (m, 1H), 2.29-2.19 (m, 4H), 1.69-1.63 (m, 2H), 1.54-1.48 (m,2H), 1.02 (s, 1H), 0.45 (s, 2H), 0.25 (s, 2H).

Preparation of (1R, 4R)-4-(pyrrolidin-1-yl)cyclohexanamine hydrochloride(amine-49)

Crude amine-49 was synthesized in a similar manner as described for thesynthesis of amine-47 and it was obtained as a white solid.

¹H NMR (D₂O, 400 MHz) δ 3.55-3.53 (m, 3H), 3.13-2.99 (m, 4H), 2.21-2.09(m, 2H), 2.06-1.99 (m, 7H), 1.49-1.40 (m, 4H).

Preparation of4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)-N-((1R,4R)-4-(pyrrolidin-1-yl)cyclohexyl)pyrimidin-2-amine (A-49)

Compound A-49 was prepared according to the procedure described inmethod F with amine-49 and it was obtained as a white solid afterpurification by prep HPLC (HCl condition). Yield: 89.3%

LCMS: RT=2.607 min, m/z 381.3 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.16 (s, 1H), 8.01 (s, 1H), 7.19 (s, 1H), 4.06(s, 1H), 3.83 (s, 3H), 3.08 (s, 2H), 2.26-1.48 (m, 13H), 1.09 (s, 1H),0.45 (s, 2H), 0.25 (s, 2H).

Preparation of (1R, 4R)-4-(azetidin-1-yl)cyclohexanamine hydrochloride(amine-50)

Crude amine-50 was synthesized in a similar manner as described for thesynthesis of amine-47 and it was obtained as a white solid

¹H NMR (CDCl₃, 400 MHz) δ 4.15-4.08 (m, 2H), 3.65-3.12 (m, 1H),2.28-2.03 (m, 6H), 1.50-1.42 (m, 4H), 1.26-1.20 (m, 1H).

Preparation of N-(1R,4R)-4-(azetidin-1-yl)cyclohexyl)-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine(A-50)

Compound A-50 was prepared according to the procedure described inmethod F with amine-50 and it was obtained as a white solid afterpurification by prep HPLC (Basic condition). Yield: 16%

LCMS: RT=2.494 min, m/z 367.3 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) 8.17 (d, J=6.8 Hz, 1H), 7.88 (s, 1H), 6.70 (d,J=6.8 Hz, 1H), 4.86 (d, J=11.6 Hz, 1H), 3.91 (m, 4H), 3.25-3.20 (m, 5H),2.31-2.07 (m, 6H), 2.05-2.02 (m, 2H), 1.24-1.10 (m, 5H), 0.47-0.43 (m,2H), 0.25-0.22 (s, 2H).

Preparation of (1R,4R)—N¹-(4-(5-(cyclobutylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-45)

Step 1: A solution of 2-cyclobutylacetonitrile (1.00 g, 10.51 mmol, 1.00eq) in HCl (6 M, 10.00 mL, 5.71 eq) was stirred at 120° C. for 16 h. Themixture was diluted with water (50 mL) and extracted with EA (20 mL*2).The combined organic layer was washed with water (40 mL), dried andconcentrated to give 45_1 (750 mg, 6.57 mmol, 62.5% yield) as acolorless liquid

¹H NMR (CDCl₃, 400 MHz) δ 2.72-2.68 (m, 1H), 2.47-2.45 (d, J=8.0 Hz,2H), 2.17-2.15 (m, 2H), 1.90-1.88 (m, 2H), 1.75-1.70 (m, 2H).

Step 2-4: Compound 45_5 was synthesized in a similar manner as describedfor the synthesis of Core A in scheme 1.1.

Compound 45_2 was obtained as a colorless liquid.

¹H NMR (CDCl₃, 400 MHz) δ 3.68 (s, 3H), 3.16 (s, 3H), 3.75-3.71 (m, 1H),2.55-2.53 (m, 2H), 2.16-2.13 (m, 3H), 1.89-1.87 (m, 2H), 1.73-1.68 (m,2H).

Compound 45_4 was obtained as a yellow oil

LCMS: RT=0.795 min, m/z 237.1 [M+H]⁺

Compound 45_5 was obtained as a dark brown solid:

¹H NMR (CDCl₃, 400 MHz) δ 8.39-8.38 (d, J=4.8 Hz, 1H), 8.02 (s, 1H),7.59 (s, 1H), 6.96-6.92 (m, 1H), 2.96 (s, 3H), 2.89 (m, 5H), 2.72-2.68(m, 1H), 2.57 (s, 3H), 2.10-2.05 (m, 2H), 1.87-1.82 (m, 4H).

Step 5-6: Compound 45_7 was synthesized in a similar manner as describedfor the synthesis of Core C in scheme 1.2.

Compound 45_6 was obtained as a yellow oil after purification by prepHPLC (TFA condition). Yield: 21.2%;

¹H NMR (CDCl₃, 400 MHz) δ 8.41-8.39 (d, J=7.2 Hz, 1H), 7.88 (s, 1H),7.09-7.07 (d, J=6.8 Hz, 1H), 3.89 (s, 3H), 3.35-3.33 (d, J=9.6 Hz, 2H),2.72-2.67 (m, 1H), 2.6 (s, 3H), 2.02-1.98 (m, 2H), 1.84-1.76 (m, 4H).

Compound 45_7 was obtained as a yellow oil after purification bypre-TLC. Yield: 86.6%;

LCMS: RT=0.706 min, m/z 307.2 [M+H]⁺

Step 7: Compound A-45 was made according to the procedure described inmethod D with trans-cyclohexane-1,4-diamine (4.0 eq) and it was obtainedas a yellow solid after purification by prep HPLC (basic condition).Yield: 21.9%;

LCMS: RT=0.539 min, m/z 341.3 [M+H]⁺;

¹H NMR (CDCl₃, 400 MHz) δ 8.18-8.17 (d, J=5.2 Hz, 1H), 7.82 (s, 1H),6.69-6.68 (d, J=5.2 Hz, 1H), 4.85-4.83 (d, J=8.0 Hz, 1H), 3.91-3.84 (m,4H), 3.33-3.31 (d, J=7.6 Hz, 2H), 2.75-2.68 (m, 2H), 2.18-2.17 (m, 2H),2.00-1.93 (m, 4H), 1.80-1.77 (m, 4H), 1.31-1.26 (m, 4H).

Preparation of (1R,4R)—N¹-(4-(5-(cyclopentylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-46)

Compound A-46 was synthesized in a manner similar as described for thesynthesis of compound A-45.

Compound 46_2 was obtained as a colorless oil.

¹H NMR (CDCl₃, 400 MHz) δ 3.64 (s, 3H), 3.14 (s, 3H), 2.41-2.40 (t,J=7.2 Hz, 2H), 2.26-2.25 (m, 1H), 1.82-1.80 (m, 2H), 1.59-1.51 (m, 4H),1.15-1.13 (m, 2H).

Compound 46_5 was obtained as a yellow oil.

LCMS: RT=1.294 min, m/z 306.2 [M+H]⁺

Compound 46_6 was obtained as a yellow oil after purification by prepTLC. Yield: 26.5%

LCMS: RT=0.866 min, m/z 289.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.42-8.41 (d, J=5.2 Hz, 1H), 7.90 (s, 1H),7.10-7.09 (d, J=5.2 Hz, 1H), 3.90 (s, 3H), 3.26-3.24 (d, J=7.6 Hz, 2H),2.61-2.60 (d, J=4.8 Hz, 3H), 2.21 (m, 1H), 1.72-1.67 (m, 4H), 1.27-1.26(m, 2H), 1.25 (m, 3H).

Compound 46_7 was obtained as a white solid after purification by prepTLC. Yield: 44.0%

LCMS: RT=1.219 min, m/z 321.2 [M+H]⁺

Compound A-46 was obtained as a yellow solid after purification by prepHPLC (basic condition). Yield: 52.7%

LCMS: RT=2.381 min, m/z 355.2 [M+H]⁺

¹H NMR (CD₃OD, 400 MHz) δ 8.16 (s, 1H), 8.07-8.03 (t, J=8.0 Hz, 1H),7.20-7.18 (d, J=6.8 Hz, 1H), 4.06-4.02 (m, 1H), 3.88 (s, 3H), 3.31-3.30(m, 2H), 3.16 (s, 1H), 2.17-2.15 (m, 5H), 1.67 (m, 4H), 1.56 (m, 6H),1.30-1.25 (m, 2H)

Preparation of (1R,4R)—N′-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-51)

Step 1: To a solution of compound N-methylpyrazole (51_1, 8.00 g, 97.44mmol, 1.00 eq) in THF (160 mL) was added drop-wise n-BuLi (2.5 M, 46.77mL, 1.20 eq) at −78° C. After the resulting mixture was stirred for 1 hat that temperature, the solution of cyclopropanecarbaldehyde (8.20 g,116.93 mmol, 1.20 eq) in THF (80 mL) was added drop-wise. And then thereaction mixture was stirred at 20° C. for 16 h. TLC (PE:EA=2:1) showedreactant 1 (Rf=0.3) was consumed and product (Rf=0.05) was formed. Themixture was poured into aqueous NH₄Cl (300 mL) and stirred for 10 min.The aqueous phase was extracted with ethyl acetate (100 mL*2). Thecombined organic phase was washed with brine (100 mL), dried overanhydrous Na₂SO₄, filtered and concentrated in vacuum. The residue waspurified by column chromatography (SiO₂, PE:EA=8:1˜0:1) to affordcompound 51_3 (12.00 g, 78.85 mmol, 80.9% yield, 100% purity) as acolorless oil.

LCMS: RT=0.118 min, m/z 153.1 [M+H]⁺

Step 2: The reaction mixture of compound 51_3 (9.00 g, 59.14 mmol, 1.00eq), TFA (40.46 g, 354.84 mmol, 26.27 mL, 6.00 eq) and Et₃SiH (41.26 g,354.84 mmol, 56.52 mL, 6.00 eq) in DCM (900 mL) was stirred at 40° C.for 36 h. The mixture was adjusted to pH=8 with aqueous NaHCO₃ andseparated. The organic layer was concentrated and purified by prep HPLC(basic condition) to give compound 51_5 (2.10 g, 15.42 mmol, 26.1%yield) as a dark brown oil.

LCMS: RT=0.565 min, m/z 137.1 [M+H]⁺

Step 3: To a solution of compound 515 (2.10 g, 15.42 mmol, 1.00 eq) inDCM (21 mL) was added NBS (3.02 g, 16.96 mmol, 1.10 eq) at 0° C. Themixture was stirred at 20° C. for 2 h. The mixture was concentrated andpurified by column chromatography (SiO₂, PE:EA=20:1) to give compound51_6 (3.00 g, 13.95 mmol, 90.5% yield) as a yellow oil.

LCMS: RT=0.784 min, m/z 217.1 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 7.39 (s, 1H), 3.87 (s, 3H), 2.65-2.63 (d,J=8.8 Hz, 2H), 0.98-0.94 (m, 1H), 0.55-0.51 (m, 2H), 0.29-0.25 (m, 2H).

Step 4: To a solution of compound 51_6 (3.00 g, 13.95 mmol, 1.00 eq) inTHF (60 mL) was added n-BuLi (2 M, 10.46 mL, 1.50 eq) drop-wise at −78°C. After stirring for 0.5 h at that temperature, a solution of2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5.19 g, 27.90mmol, 2.00 eq) in THF (6 mL) was added. The resulting mixture was warmedto 20° C. and stirred for 0.5 h. TLC (PE:EA=5:1) showed reactant(Rf=0.6) was consumed and product (Rf=0.5) was formed. The mixture wasquenched with saturated NH₄Cl (50 mL) and extracted with EA (100 mL).The organic layer was concentrated and purified by column chromatography(SiO₂, PE:EA=20:1˜10:1) to give compound 51_7 (3.30 g, 11.40 mmol, 81.7%yield, 90.5% purity) as a colorless oil.

LCMS: RT=0.801 min, m/z 263.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 7.67 (s, 1H), 3.85 (s, 3H), 2.82-2.81 (d,J=6.8 Hz, 2H), 1.30 (s, 12H), 0.92-0.90 (m, 1H), 0.45-0.42 (m, 2H),0.29-0.27 (m, 2H).

Step 5: To a solution of compound 51_7 (500.00 mg, 1.91 mmol, 1.00 eq)in DME (10 mL) were added 2,4,5-trichloropyrimidine (51_8, 420.40 mg,2.29 mmol, 1.20 eq), Na₂CO₃ (2 M, 2.10 mL, 2.20 eq) and catalystPdCl₂(Amphos)₂ (67.62 mg, 95.50 μmol, 0.05 eq) under nitrogen. Theresulting mixture was stirred at 85° C. for 2 hr under nitrogen. TLC(PE:EA=5:1) showed reactant (Rf=0.4) was consumed and product (Rf=0.5)was formed. The mixture was diluted with water (50 mL) and extractedwith EA (50 ml). The organic layer was concentrated and purified bysilica gel column (PE:EA=5:1) to afford compound 519 (350.0 mg, 1.05mmol, 55% yield, 85% purity) as a yellow oil.

LCMS: RT=0.819 min, m/z 283.1 [M+H]⁺

Step 6: The reaction mixture of compound 51_9 (300.00 mg, 1.06 mmol,1.00 eq) and trans-cyclohexane-1,4-diamine (484.17 mg, 4.24 mmol, 4.00eq) in dioxane (4.5 mL) was stirred at 130° C. for 2 h under microwave.The mixture was filtered and concentrated. The crude was purified byprep-HPLC (HCl condition) to give A-51 (80.00 mg, 200.04 μmol, 18.9%yield, 99.3% purity, HCl) as a yellow solid.

LCMS: RT=2.817 min, m/z 361.1 [M+H]⁺

¹H NMR (DMSO-d, 400 MHz) δ 8.37 (s, 1H), 8.29 (s, 3H), 8.08 (s, 1H),3.86 (s, 3H), 3.70-3.68 (m, 2H), 3.06-3.05 (d, J=6.4 Hz, 2H), 2.96 (s,1H), 2.03-1.95 (m, 4H), 1.49-1.32 (m, 4H), 0.98 (s, 1H), 0.42-0.40 (m,2H), 0.16 (m, 2H).

Preparation of (1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)-5-methylpyrimidin-2-yl)cyclohexane-1,4-diamine(A-52)

Step 1: To a solution of compound 51_7 (500.00 mg, 1.91 mmol, 1.00 eq)in DME (10 mL) were added compound 52_8 (373.60 mg, 2.29 mmol, 1.20 eq),Na₂CO₃ (a.q) (2 M, 2.1 mL, 2.20 eq) and catalyst PdCl₂(Amphos)₂ (67.62mg, 95.50 μmol, 0.05 eq) under nitrogen. The resulting mixture wasstirred at 85° C. for 2 h under nitrogen. TLC (PE:EA=5:1) showedreactant (Rf=0.4) was consumed and product (Rf=0.25) was formed. Themixture was diluted with water (50 mL) and extracted with EA (50 mL).The organic layer was concentrated and purified by column chromatography(SiO₂, PE:EA=20:1-5:1) to give compound 529 (350.00 mg, 1.15 mmol, 60.3%yield, 86.5% purity) as a yellow oil.

LCMS: RT=0.761 min, m/z 263.2 [M+H]⁺

Step 2: The reaction mixture of compound 52_9 (350.00 mg, 1.33 mmol,1.00 eq) and trans-cyclohexane-1,4-diamine (607.49 mg, 5.32 mmol, 4.00eq) in dioxane (5 mL) was stirred at 130° C. for 2 h under microwave.TLC (PE:EA=1:1) showed reactant (Rf=0.6) was consumed and product(Rf=0.05) was formed. The mixture was filtered and concentrated. Thecrude was purified by prep-HPLC (HCl condition) and then prep-HPLC(basic condition) to give A-52 (30.0 mg, 88.00 μmol, 6.6% yield, 99.8%purity) as a yellow solid.

LCMS: RT=2.429 min, m/z 341.2 [M+H]⁺

¹H NMR (CDCl3, 400 MHz) 8.09 (s, 1H), 7.67 (s, 1H), 4.72-4.70 (d, J=8.4Hz, 1H), 3.91 (s, 3H), 3.82-3.80 (m, 1H), 3.02-3.00 (d, J=6.4 Hz, 2H),2.79-2.76 (m, 1H), 2.22 (s, 3H), 2.14-1.93 (m, 4H), 1.32-1.23 (m, 4H),0.97 (m, 1H), 0.45-0.42 (m, 2H), 0.12-0.10 (m, 2H).

Preparation of(1R,4R)—N¹-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-H-pyrazol-4-yl)pyrimidin-2-yl)-4-methylcyclohexane-L4-diamine(A-30_1) and(1S,4S)—N1-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-4-methylcyclohexane-1,4-diamine(A-30_2)

Step 1: To a mixture of compound 30_1 (900.00 mg, 3.96 mmol, 1.00 eq)and BnNH₂ (424.26 mg, 3.96 mmol, 432.92 μL, 1.00 eq) in DCM (18.00 mL)were added AcOH (237.77 mg, 3.96 mmol, 226.45 μL, 1.00 eq) andNaBH(OAc)₃ (1.68 g, 7.92 mmol, 2.00 eq). The resulting mixture wasstirred at 20° C. for 2 hours. The mixture was quenched with H₂O (50 mL)and separated. The organic layer was concentrated. The residue waspurified by column chromatography on Al₂O₃ (DCM:MeOH=20:1) to givecompound 30_2 (650.00 mg, 2.03 mmol, 51.3% yield, 99.6% purity) as a redoil.

LCMS: RT=1.520 min, m/z 319.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 7.33-7.31 (m, 5H), 4.43-4.35 (m, 1H), 3.81 (d,J=10.8 Hz, 2H), 2.62-2.50 (m, 1H), 2.12-2.09 (m, 1H), 1.82-1.79 (m, 4H),1.43 (s, 9H), 1.38-1.27 (m, 7H).

Step 2: To a solution of compound 30_2 (350.00 mg, 1.10 mmol, 1.00 eq)in MeOH (3.5 mL) was added Pd(OH)₂ (35.00 mg). The mixture was stirredat 20° C. for 16 hours under H₂ (16 psi). TLC (DCM:MeOH=10:1) showedreactant (Rf=0.6) consumed and product (Rf=0.3) formed. The mixture wasfiltered and the mother liquid was concentrated to give amine-30 (220.00mg, 963.5 umol, 87.6% yield) as a red oil.

MS: m/z 229.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 4.43-4.33 (m, 1H), 3.82-2.66 (m, 1H), 2.08 (s,1H), 1.79-1.66 (m, 4H), 1.43 (s, 9H), 1.33-1.27 (m, 7H).

Step 3: To a solution of Amine-30 (300.00 mg, 917.99 μmol, 1.00 eq) andCore D (230.57 mg, 1.01 mmol, 1.10 eq) in DMSO (4 mL) were added DIEA(480.97 μL, 2.75 mmol, 3.00 eq) and TBAF (48.00 mg, 2.75 mmol, 0.20 eq).The mixture was stirred at 160° C. for 3 hours. The mixture wasseparated between EA (50 mL) and H₂O (50 mL). The organic layer wasconcentrated to give 30_3 (400.00 mg, crude) as a red oil.

LCMS: RT=1.081 min, m/z 475.2 [M+H]⁺

Step 4: To a solution of 30_3 (400.00 mg, 842.05 μmol, 1.00 eq) in DCM(4 mL) was added TFA (800 μL) at 0° C. The resulting mixture was stirredat 20° C. for 1 hour. TLC (PE:EA=2:1) showed reactant (Rf=0.6) consumedand product (Rf=0.05) formed. The mixture was concentrated. The residuewas purified by prep-HPLC (HCl condition) to give A-30_1 (55.00 mg,127.60 μmol, 15.1% yield, 95.4% purity, HCl) and A-30_2 (40.00 mg, 95.31μmol, 11.3% yield, 98.0% purity, HCl) as yellow solid.

LCMS (A-30_1): RT=2.495 min, m/z 375.2 [M+H]⁺

¹H NMR_Peak-1 (MeOD, 400 MHz) δ 8.49 (s, 1H), 8.45 (s, 1H), 4.10 (s,1H), 3.96 (s, 3H), 3.23 (d, J=6.4 Hz, 2H), 2.11-2.10 (m, 2H), 2.07-1.94(m, 2H), 1.84-1.74 (m, 4H), 1.47 (s, 3H), 1.09-1.08 (m, 1H), 0.55-0.51(m, 2H), 0.28-0.27 (m, 2H).

LCMS (A-30_2): RT=2.527 min, m/z 375.2 [M+H]⁺

¹H NMR_Peak-2 (MeOD, 400 MHz) δ 8.47 (s, 2H), 4.19 (s, 1H), 3.96 (s,3H), 3.19 (d, J=6.4 Hz, 2H), 2.05-2.1.99 (m, 2H), 1.87-1.78 (m, 2H),1.42 (s, H), 1.08-1.07 (m, 1H), 0.54-0.52 (m, 2H), 0.30-0.28 (m, 2H).

Preparation of(1R,4R)—N¹-(4-(5-(cyclobutylmethyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A53)

Step 1: To a mixture of NaCN (13.81 g, 281.82 mmol, 1.40 eq) in DMSO(240 mL) was added 53_1 (30.00 g, 201.30 mmol, 22.56 mL, 1.00 eq)dropwise at 60° C. The mixture was kept at 75° C. for 16 hr. The mixturewas cool down and diluted with water (500 mL). The solution wasextracted with EtOAc (200 mL*3). The combined organic layers were washedwith brine (100 mL*3) and dried. The organic layer was concentrated togive 53_2 (15.00 g, 157.66 mmol, 78.3% yield) as a light yellow liquid.

¹H NMR (CDCl₃, 400 MHz) δ 2.64-2.63 (m, 1H), 2.42-2.41 (d, J=6.4 Hz,2H), 2.20-2.17 (m, 2H), 1.90-1.83 (m, 4H).

Step 2: A solution of 53_2 (15.00 g, 157.66 mmol, 1.00 eq) in HCl (6 M,150 mL, 5.71 eq) was stirred at 120° C. for 16 hours. The mixture wasdiluted with water (500 ML) and extracted with EtOAc (200 mL*2). Thecombined organic layers were washed with water (400 mL). The organiclayer was dried and concentrated to give 53_3 (16.00 g, 140.18 mmol,88.9% yield) as a colorless liquid.

¹H NMR (CDCl₃, 400 MHz) δ 2.69-2.68 (m, 1H), 2.47-2.45 (m, 2H),2.17-2.15 (m, 2H), 1.89-1.87 (m, 2H), 1.75-1.70 (m, 2H).

Step 3: To a solution of 53_3 (16.00 g, 140.18 mmol, 1.00 eq) andN-methoxymethanamine (20.51 g, 210.27 mmol, 1.50 eq, HCl) in DCM (160mL) was added CDI (45.46 g, 280.36 mmol, 2.00 eq) at 0° C. in portions.The mixture was stirred at 20° C. for 16 hours. The mixture was dilutedwith water (50 mL) and extracted with DCM (30 mL*3). The combinedorganic layer was dried and concentrated to give crude product. Thecrude product was purified by silica gel column (PE:EA=50:1-10:1) togive 53_4 (12.00 g, 76.33 mmol, 54.4% yield) as a colorless liquid.

¹H NMR (CDCl₃, 400 MHz) δ 3.68-3.67 (d, J=2.4 Hz, 3H), 3.16 (s, 3H),2.77-2.73 (m, 1H), 2.54-2.53 (m, 2H), 2.16-2.13 (m, 2H), 1.88-1.86 (m,2H), 1.73-1.72 (m, 2H), 1.70-1.68 (m, 2H).

Step 4: To a solution of 53_4 (4.50 g, 32.09 mmol, 1.00 eq) in THF (225mL) was added LDA (2 M, 24.07 mL, 1.50 eq) at −78° C. After stirred for1 hr, a solution of 2-cyclobutyl-N-methoxy-N-methyl-acetamide (6.05 g,38.51 mmol, 1.20 eq) in THF (120 mL) was added into it at −78° C. Theresulting mixture was stirred at −78° C. for 4 hr, quenched withsaturated NH₄Cl (200 mL), and the aqueous phase was extracted with ethylacetate (200 mL*3). The combined layers were washed with brine (200 mL),dried over anhydrous Na₂SO₄, filtered and concentrated to give 53_5(10.00 g, crude) as a yellow oil.

LCMS: RT=0.788 min, m/z 237.1 [M+H]⁺

Step 5: The solution of 53_5 (10.00 g, 42.31 mmol, 1.00 eq) in DMF-DMA(201.68 g, 1.69 mol, 224.09 mL, 40.00 eq) was stirred at 90° C. for 2hr. The mixture was concentrated to give crude product. The crudeproduct was purified by silica gel column (DCM:MeOH=1:0-10:1) to give53_6 (8.80 g, crude) as a black brown solid.

Step 6: To a solution of 53_6 (800.00 mg, 2.75 mmol, 1.00 eq) in EtOH(12 mL) was added isopropylhydrazine (364.95 mg, 3.30 mmol, 1.20 eq,HCl) and TEA (333.93 mg, 3.30 mmol, 457.43 uL, 1.20 eq). The mixture wasstirred at 90° C. for 1 hr. The mixture was concentrated. The crude waspurified by pre-HPLC (TFA) to give 53_7 (600.00 mg, 1.88 mmol, 68.5%yield, 95% purity) as a yellow oil.

LCMS: RT=0.901 min, m/z 303.2 [M+H]⁺

Step 7: To a solution of 53_7 (600.00 mg, 1.98 mmol, 1.00 eq) in DCM (9mL) was added m-CPBA (1.01 g, 4.96 mmol, 85% purity, 2.50 eq) at 0° C.The mixture was stirred at 20° C. for 3 hr. The reaction was quenchedwith saturated a.q NaS₂O₃ (50 mL) and extracted with DCM (20 mL*2). Thecombined organic layers were washed with a.q NaHCO₃ (40 mL). The organiclayer was dried and concentrated. The crude was purified by prep-TLC(DCM:MeOH=10:1)(Rf=0.6) to give 538 (500.00 mg, 1.50 mmol, 75.5% yield)as a yellow oil.

LCMS: RT=0.803 min, m/z 335.1 [M+H]⁺

Step 8: To a solution of 53_8 (500.00 mg, 1.50 mmol, 1.00 eq) in dioxane(7.5 mL) was added trans-cyclohexane-1,4-diamine (685.14 mg, 6.00 mmol,4.00 eq). The mixture was stirred at 130° C. for 2 hr in the microwave.The mixture was filtered and concentrated. The crude was purified byprep HPLC (base) and prep HPLC(HCl) to give A-53 (250.00 mg, 615.9 μmol,41% yield, 99.8% purity, HCl) as a yellow solid.

LCMS: RT=2.57 min, m/z 369.2 [M+H]⁺

¹H NMR (DMSO, 400 MHz) δ 8.88-8.87 (m, 1H), 8.38-8.25 (m, 5H), 7.30-7.28(m, 1H), 4.78-4.76 (m, 1H), 3.36-3.34 (m, 2H), 3.05 (s, 1H), 2.61 (s,1H), 2.05 (m, 4H), 1.92-1.91 (m, 2H), 1.78 (m, 4H), 1.47-1.39 (m, 10H).

Preparation of(1R,4R)—N-(4-(1-methyl-5-((1-methylcyclopropyl)methyl)-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A56)

Step 1: To a solution of 1-methylcyclopropane-1-carboxylic acid (4.00 g,39.95 mmol, 1.00 eq) and CDI (7.13 g, 43.95 mmol, 1.10 eq) in DCM (40mL) was added N-methoxymethanamine (4.68 g, 47.94 mmol, 1.20 eq, HCl) at0° C. in portions. The mixture was stirred at 20° C. for 16 hr. Themixture was diluted with water (50 mL) and extracted with DCM (20 mL*3).The combined organic layers were dried and concentrated to give crudeproduct. The crude product was purified by silica gel column(PE:EA=30:1-10:1) to give 56_1 (3.30 g, 23.05 mmol, 57.7% yield) as acolorless liquid.

¹H NMR (CDCl₃, 400 MHz) δ 3.73 (s, 3H), 3.23 (s, 3H), 1.37 (s, 3H),1.05-1.03 (m, 2H), 0.58-0.55 (m, 2H).

Step 2: To a solution of 1-methylpyrazole (1.70 g, 20.71 mmol, 1.72 mL,1.00 eq) in THF (35.00 mL) was added n-BuLi (2.5 M, 9.94 mL, 1.20 eq) at−78° C. Stirred for 1 hr. Compound 56_1 (3.26 g, 22.78 mmol, 1.10 eq) inTHF (35 mL) was added to it at −78° C. The resulting mixture was stirredat 20° C. for 1 hr. The mixture was quenched with saturated NH₄Cl (20mL) and extracted with EtOAc (20 mL*2). The organic layer wasconcentrated and purified by silica gel column (PE:EA=1:0-20:1) to give56_2 (2.50 g, 13.41 mmol, 64.8% yield, 88.1% purity) as a yellow oil.

LCMS: RT=0.609 min, m/z 165.1 [M+H]⁺

Step 3: The reaction mixture of 56_2 (2.00 g, 12.18 mmol, 1.00 eq) andKOH (2.73 g, 48.72 mmol, 4.00 eq) in NH₂NH₂.H₂O (2.57 g, 48.72 mmol,2.49 mL, 95% purity, 4.00 eq) and diglycol (40 mL) was heated to 110° C.for 1.5 hr, then at 200° C. for another 1 hour with Dean-Stark. Themixture was diluted with water (50 mL) and extracted with MTBE (50mL*2). The combined organic layers were concentrated to give 56_3 (1.10g, crude) as a colorless oil.

LCMS: RT=0.639 min, m/z 151.1 [M+H]⁺

Step 4: To a solution of 563 (1.10 g, 7.32 mmol, 1.00 eq) in DCM (11 ML)was added NBS (1.30 g, 7.32 mmol, 1.00 eq) at 0° C. The mixture wasstirred at 20° C. for 1 hr. The mixture was concentrated. The crude waspurified by silica gel column (PE:EA=1:0-50:1) to give 56_4 (1.45 g,5.65 mmol, 77.1% yield, 89.2% purity) as a colorless oil.

LCMS: RT=0.835 min, m/z 229.0 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 7.39 (s, 1H), 3.85 (s, 3H), 2.73 (s, 2H), 1.05(s, 3H), 0.44-0.31 (m, 4H).

Step 5: To a solution of 56_4 (1.45 g, 6.33 mmol, 1.00 eq) in THF (29mL) was added n-BuLi (2 M, 4.75 mL, 1.50 eq) at −78° C. After 30 min,2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.36 g, 12.66mmol, 2.59 mL, 2.00 eq) in THF (2.5 ML) was added to it. The resultingmixture was warmed to 20° C. and stirred for 0.5 hr. The mixture wasquenched with saturated NH₄Cl (50 mL) and extracted with EtOAc (100 mL).The organic layer was concentrated and purified by silica gel column(PE:EA=20:1˜10:1) to give 56_5 (1.25 g, 4.11 mmol, 64.9% yield, 90.7%purity) as a colorless oil.

LCMS: RT=0.928 min, m/z 277.1 [M+H]⁺

Step 6: To a solution of 56_5 (500.00 mg, 1.81 mmol, 1.00 eq) in DME (10mL) was added 4-chloro-2-methylsulfanyl-pyrimidine (290.79 mg, 1.81mmol, 210.72 μL, 1.00 eq), NA2CO3 (2 M, 1.99 mL, 2.20 eq) and4-ditert-butylphosphanyl-N,N-dimethyl-aniline; dichloropalladium (64.09mg, 90.50 μmol, 64.09 μL, 0.05 eq) under nitrogen. The resulting mixturewas stirred at 85° C. for 2 hr under nitrogen. The mixture was dilutedwith water (50 mL) and extracted with EtOAc (50 mL). The organic layerwas concentrated and purified by silica gel column (PE:EA=5:1) to give56_6 (350.00 mg, 1.14 mmol, 63% yield, 89% purity) as a yellow oil.

LCMS: RT=0.829 min, m/z 275.1 [M+H]⁺

Step 7: To a solution of 56_6 (350.00 mg, 1.28 mmol, 1.00 eq) in DCM(5.5 mL) was added MCPBA (690.28 mg, 3.20 mmol, 80% purity, 2.50 eq) at0° C. The mixture was stirred at 20° C. for 2 hr. The mixture wasquenched with aqueous Na₂S₂O₃ (100 mL) and extracted with DCM (50 mL*2).The combined organic layers were concentrated and purified by silica gelcolumn (PE:EA=1:1) to give 56_7 (200.00 mg, 617.72 μmol, 48.6% yield,94.6% purity) as a yellow solid.

LCMS: RT=0.653 min, m/z 307.1 [M+H]⁺

Step 8: The mixture of 56_7 (200.00 mg, 652.78 μmol, 1.00 eq) andtrans-cyclohexane-1,4-diamine (298.17 mg, 2.61 mmol, 4.00 eq) in dioxane(3 mL) was stirred at 130° C. for 2 hr in the microwave. The mixture wasfiltered and concentrated. The crude was purified by prep-HPLC (base) togive A-56 (50.00 mg, 144.85 μmol, 22.2% yield, 98.6% purity) as a yellowsolid.

LCMS: RT=1.999 min, m/z 341.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.18-8.17 (d, J=5.2 Hz, 1H), 7.82 (s, 1H),6.69-6.67 (d, J=5.2 Hz, 1H), 4.87-4.85 (d, J=5.6 Hz, 1H), 3.88 (s, 3H),3.84 (s, 1H), 3.38 (s, 2H), 2.75-2.73 (m, 1H), 2.17-2.16 (m, 2H),1.94-1.92 (m, 2H), 1.31-1.27 (m, 4H), 1.08 (s, 3H), 0.31-0.25 (m, 4H).

Preparation of(1R,4R)—N¹-(4-(1-methyl-5-neopentyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A57)

Compound A-57 was synthesized in a manner similar as described for thesynthesis of compound A-56.

LCMS: RT=1.558 min, m/z 343.2 [M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.20-8.13 (m, 2H), 7.29-7.27 (d, J=5.2 Hz, 1H),4.12-4.09 (m, 1H), 3.92 (s, 3H), 3.34 (s, 1H), 3.21 (s, 1H), 2.20 (m,4H), 1.63 (m, 4H), 0.98 (s, 9H).

Preparation of(4-(2-(((1R,4R)-4-aminocyclohexyl)amino)pyrimidin-4-yl)-1-methyl-1H-pyrazol-5-yl)(cyclopropyl)methanol(A58)

Step 1 to Step 6: Intermediate 58_6 was synthesized from compound 513 ina manner similar as described for the synthesis of A-56.

LCMS: RT=0.676 min, m/z 457.4 [M+H]⁺

Step 7: To a solution of 58_6 (300.00 mg, 656.89 μmol, 1.00 eq) in THF(300 μL) was added TBAF.3H₂O (414.51 mg, 1.31 mmol, 2.00 eq) at 20° C.The mixture was stirred for 30 min. The mixture was filtered andconcentrated. The crude product was purified by prep-HPLC (base) to giveA-58 (27.00 mg, 76.89 μmol, 11.7% yield, 97.5% purity) as a yellowsolid.

LCMS: RT=2.29 min, m/z 343.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.23-8.22 (d, J=5.2 Hz, 1H), 7.86 (s, 1H),6.81-6.80 (d, J=5.2 Hz, 1H), 5.09 (s, 1H), 4.32-4.30 (m, 1H), 3.89 (s,3H), 3.81-3.79 (m, 1H), 2.93-2.88 (m, 1H), 2.20-2.06 (m, 4H), 1.52-1.31(m, 5H), 0.59-0.28 (m, 4H).

Preparation of(1R,4R)—N¹-(5-chloro-4-(5-(cyclopropylmethyl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A59)

Compound A-59 was synthesized in a manner similar as described for thesynthesis of compound A-51.

LCMS: RT=2.478 min, m/z 389.2 [M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.42-8.36 (m, 2H), 4.82-4.75 (m, 1H), 4.00 (s,1H), 3.21-3.20 (s, 3H), 2.20-2.15 (m, 4H), 1.60-1.56 (m, 4H), 1.51 (s,6H), 1.05 (s, 1H), 0.55-0.53 (m, 2H), 0.28 (m, 2H).

Preparation of(1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)-5-(trifluoromethyl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A60)

Step 1: To a solution of 60_1 (2.00 g, 9.22 mmol, 1.00 eq) in THF (40ML) was added ZnCl₂-Et₂O (1 M, 11.06 mL, 1.20 eq) at 0° C. undernitrogen protection. The mixture was stirred for 2 hours at 0° C.Methylsulfanylsodium (646.23 mg, 9.22 mmol, 587.48 μL, 1.00 eq) wasadded. The resulting mixture was stirred at 20° C. for 16 hours. TLC(pure PE) showed reactant 1 (Rf=0.5) consumed and product (Rf=0.3)formed. The mixture was quenched with 1M HCl (20 mL) and concentrated.The aqueous layer was extracted with DCM (20 mL*3). The combined organiclayer was concentrated and purified by silica gel column(PE:EA=1:0˜50:1) to give 60_2 (1.00 g, 1.97 mmol, 21.4% yield, 45.1%purity) as colorless oil.

LCMS: RT=0.794 min, m/z 228.9 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.67 (s, 1H), 2.62 (s, 3H).

Step 2: To a solution of 51_7 (574.11 mg, 2.19 mmol, 1.00 eq) in THF (10mL) were added 60_2 (500.00 mg, 2.19 mmol, 1.00 eq), K₃PO₄ (1 M, 4.38mL, 2.00 eq) and Ad₂nBuP. Biphenyl (50.00 mg) under nitrogen. Theresulting mixture was stirred at 85° C. for 2 hours under nitrogen. TLC(PE:EA=1:1) showed reactant (Rf=0.6) consumed and product (R=0.5)formed. The mixture was concentrated and diluted with H₂O (50 mL). Theaqueous layer was extracted with EA (20 mL*2). The organic layer wasconcentrated. The residue was purified by silica gel column(PE:EA=20:1-5:1) to give 60_3 (300.00 mg, 611.96 umol, 27.9% yield,66.9% purity) as a yellow solid.

LCMS: RT=0.927 min, m/z 329.0 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.75 (s, 1H), 7.75 (s, 1H), 3.94 (s, 3H), 2.95(d, J=6.8 Hz, 2H), 2.57 (s, 3H), 0.98-0.94 (m, 1H), 0.48-0.44 (m, 2H),0.17-0.13 (m, 2H).

Step 3: To a solution of 60_3 (400.00 mg, 1.22 mmol, 1.00 eq) in DCM (7mL) was added m-CPBA (657.92 mg, 3.05 mmol, 80% purity, 2.50 eq) at 0°C. The mixture was stirred at 20° C. for 1 hour. TLC (DCM:MeOH=20:1)showed reactant (Rf=0.6) consumed and product (Rf=0.5) formed. Themixture was quenched with a.q Na₂S₂O₃ (100 mL) and extracted with DCM(50 mL*2). The organic layer was concentrated and purified by silica gelcolumn (PE:EA=1:1) to give 60_4 (300.00 mg, 738.23 umol, 60.5% yield,88.7% purity) as a yellow solid.

LCMS: RT=0.72 min, m/z 361.0 [M+H]⁺

Step 4: The mixture of 60_4 (200.00 mg, 555.02 μmol, 1.00 eq) andtrans-cyclohexane-1,4-diamine (253.51 mg, 2.22 mmol, 4.00 eq) in dioxane(3 mL) was stirred at 130° C. for 2 hours under microwave. The mixturewas filtered and the filtrate was concentrated. The residue was purifiedby prep-HPLC (HCl condition) to give A-60 (80.00 mg, 185.66 μmol, 33.5%yield, 100% purity, HCl) as a yellow solid.

LCMS: RT=2.417 min, m/z 395.2 [M+H]⁺

¹H NMR (MeOH, 400 MHz) δ 8.66 (s, 1H), 7.90 (s, 1H), 4.15 (s, 1H), 3.97(s, 3H), 3.20 (s, 1H), 3.10 (s, 2H), 2.18 (s, 4H), 1.62 (s, 4H), 1.05(s, 1H), 0.53 (s, 2H), 0.25 (s, 2H).

Preparation ofN-((1R,4R)-4-(1H-pyrazol-1-yl)cyclohexyl)-5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine(A64)

Step 1: To a solution of 64_1 (2.00 g, 9.29 mmol, 1.00 eq) in DCM (20mL) were added TEA (1.88 g, 18.58 mmol, 2.58 mL, 2.00 eq) andmethanesulfonyl chloride (1.06 g, 9.29 mmol, 719.03 μL, 1.00 eq) at 0°C. The mixture was stirred at 20° C. for 2 hours. The mixture wasdiluted with H₂O (30 mL) and extracted with DCM (20 mL*2). The combinedorganic layers were dried and concentrated to give 64_2 (2.50 g, 8.52mmol, 91.7% yield) as a white solid.

¹H NMR (CDCl₃, 400 MHz) δ 4.88 (s, 1H), 4.48 (s, 1H), 3.52 (s, 1H), 3.01(s, 3H), 2.07-2.03 (m, 2H), 1.83-1.82 (m, 2H), 1.73-1.70 (m, 2H),1.61-1.55 (m, 7H), 1.448 (s, 9H).

Step 2: To a solution of 1H-pyrazole (195.25 mg, 2.87 mmol, 1.20 eq) inMeCN (7.00 mL) were added Cs₂CO₃ (1.56 g, 4.78 mmol, 2.00 eq) and 64_2(700.00 mg, 2.39 mmol, 1.00 eq). The mixture was stirred at 100° C. for2 hours. The mixture was diluted with H₂O (20 mL) and extracted withEtOAc (10 mL*3). The combined organic layers were washed with brine (20mL) and concentrated. The residue was purified by pre-HPLC (basiccondition) to give 64_3 (200.00 mg, 732.25 μmol, 30.6% yield, 97.1%purity) as a yellow solid.

MS: m/z RT=0.888 min, 266.0 [M+H]⁺

Step 3: To a solution of 64_3 (180.00 mg, 678.35 μmol, 1.00 eq) in DCM(2 mL) was added TFA (400.00 L) at 0° C. The mixture was stirred at 20°C. for 1 hour. The mixture was diluted with H₂O (30 mL) and extractedwith DCM (10 mL*2). The aqueous layer was lyophilized to give amine-64(180.00 mg, 633.91 μmol, 93.5% yield, 98.3% purity, TFA) as colorlessoil.

LCMS: RT=0.272 min, m/z 166.2 [M+H]⁺

Step 4: To a solution of 519 (100.00 mg, 353.16 μmol, 1.00 eq) in DMSO(1.5 mL) were added amine-64 (95.48 mg, 529.74 μmol, 1.50 eq) and DIEA(182.57 mg, 1.41 mmol, 246.71 μL, 4.00 eq). The resulting mixture wasstirred at 160° C. for 3 hours, filtered and the mother liquid wasconcentrated. The residue was purified by pre-HPLC (basic condition) andthen prep-HPLC (HCl condition) to give A-64 (10.00 mg, 23.42 μmol, 6.6%yield, 100% purity) as a yellow solid.

LCMS: RT=1.981 m/z 412.2[M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.41-8.37 (m, 2H), 7.83 (s, 1H), 7.67 (s, 1H),6.39 (s, 1H), 4.36 (s, 1H), 4.12-4.08 (m, 1H), 3.95 (s, 3H), 3.25-3.23(d, J=6.4 Hz, 2H), 2.26-2.23 (m, 4H), 2.05-2.02 (m, 2H), 1.99-1.96 (m,2H), 1.73-1.67 (m, 2H), 1.09 (s, 1H), 0.55-0.50 (m, 2H), 0.27-0.26 (m,2H).

Preparation ofN-((1R,4R)-4-(1H-imidazol-1-yl)cyclohexyl)-5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-amine(A-65)

Compound A-65 was synthesized in a manner similar as described for thesynthesis of compound A-64 as a yellow solid.

LCMS: RT=1.461 min, m/z 412.2 [M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 9.10 (s, 1H), 8.42 (s, 1H), 7.81 (s, 1H), 7.62(s, 1H), 4.55-4.49 (m, 1H), 4.13 (s, 1H), 3.96 (s, 3H), 3.25-3.24 (d,J=6.4 Hz, 2H), 2.36-2.28 (m, 4H), 2.08-1.98 (m, 2H), 1.80-1.74 (m, 2H),1.09 (s, 1H), 0.53-0.51 (m, 2H), 0.27-0.26 (m, 2H).

Preparation of(1R,4R)—N¹-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N⁴-phenylcyclohexane-1,4-diamine(A-68)

Step 1: To a solution of tert-butyl (1R,4R)-4-aminocyclohexyl)carbamate(500.00 mg, 2.33 mmol, 1.00 eq) and iodobenzene (713.01 mg, 3.49 mmol,389.63 uL, 1.50 eq) in THF (15 mL) were added t-BuOK (784.35 mg, 6.99mmol, 3.00 eq) and RuPhos Indoline (100.00 mg). The resulting mixturewas stirred at 80° C. for 12 hours under nitrogen. The mixture wasfiltered and the mother liquid was concentrated. The residue waspurified by silica gel column (PE:EA=10:1-4:1) to give 68_1 (300.00 mg,869.39 μmol, 37.3% yield, 84.1% purity) as a white solid.

LCMS: RT=1.024 min, m/z 291.0 [M+H]⁺

Step 2 and step 3: Compound A-68 was synthesized in a manner similar asdescribed for the synthesis of compound A-64 as a yellow solid.

LCMS: RT=1.194 min, m/z 437.2 [M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.29 (s, 1H), 8.03 (s, 1H), 7.75-7.34 (m, 5H),3.86 (s, 3H), 3.05 (d, J=6.4 Hz, 2H), 2.00-1.97 (m, 4H), 1.55 (s, 2H),1.37-1.29 (m, 2H), 0.97 (s, 1H), 0.39 (s, 2H), 0.14 (s, 2H).

Preparation of(5R,8R)-8-((5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)-1-azaspiro[4,5]decan-2-one(A-71)

To a solution of amine-71 (180.00 mg, 879.34 μmol, 1.00 eq, HCl) and51_9 (248.99 mg, 879.34 μmol, 1.00 eq) in DMSO (2.7 mL) were added DIEA(614.30 μL, 3.52 mmol, 4.00 eq) and TBAF (1 M, 175.87 μL, 0.20 eq). Themixture was stirred at 160° C. for 3 hours. The mixture was cooled downand filtered. The solid were washed with MeOH (10 mL) at roomtemperature and then EtOAc (3 mL) at 50° C. to give A-71 (40.00 mg,90.39 μmol, 11.4% yield, 93.7% purity) as a white solid.

LCMS: RT=2.836 min, m/z 415.2 [M+H]⁺

¹H NMR (DMSO, 400 MHz) δ 8.26 (s, 1H), 8.00 (s, 1H), 7.72 (s, 1H), 7.20(s, 1H), 3.85 (s, 3H), 3.70-3.68 (m, 1H), 3.06 (d, J=6.4 Hz, 2H),2.19-2.15 (m, 2H), 1.86-1.80 (m, 5H), 1.64-1.60 (m, 3H), 1.49-1.41 (m,5H), 0.97 (s, 1H), 0.40 (s, 2H), 0.13 (s, 2H).

Preparation of(1R,4R)—N¹-benzyl-N⁴-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-74)

To a solution of A-51 (200.00 mg, 554.20 μmol, 1.00 eq) and benzaldehyde(58.81 mg, 554.20 umol, 56.01 μL, 1.00 eq) were added AcOH (33.28 mg,554.20 μmol, 31.70 μL, 1.00 eq) and NaBH₃CN (69.65 mg, 1.11 mmol, 2.00eq). The mixture was stirred at 15° C. for 16 hours. The mixture wasquenched with aqueous NaHCO₃ (1 mL) and concentrated. The residue waspurified by prep-HPLC (column: Phenomenex Gemini 150*25 mm*10 um; mobilephase: [water (0.05% ammonia hydroxide v/v)−ACN]; B %: 55%-85%, 12 min)to give A-74 (100.00 mg, 217.80 μmol, 39.3% yield, 98.2% purity) as apink solid.

LCMS: RT=2.74 min, m/z 451.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.20-8.13 (m, 2H), 7.34-7.33 (m, 5H), 4.87 (d,J=8.4 Hz, 1H), 3.91 (s, 3H), 3.84 (s, 3H), 3.07-3.06 (m, 2H), 2.58-2.52(m, 1H), 2.16-2.02 (m, 4H), 1.32-1.22 (m, 4H), 1.01-0.99 (m, 1H),0.49-0.45 (m, 2H), 0.18-0.16 (m, 2H).

Preparation of(1R,4R)—N¹-((1H-pyrazol-4-yl)methyl)-N⁴-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-v)cyclohexane-1,4-diamine(A-75)

To a solution of A-51 (200.00 mg, 554.20 μmol, 1.00 eq) and1H-pyrazole-4-carbaldehyde (53.25 mg, 554.20 μmol, 1.00 eq) were addedAcOH (34.86 μL, 609.62 umol, 1.10 eq) and NaBH₃CN (69.65 mg, 1.11 mmol,2.00 eq). The mixture was stirred at 15° C. for 16 hours. The mixturewas quenched with aqueous NaHCO₃ (1 mL) and concentrated. The residuewas purified by prep-HPLC (column: Phenomenex Gemini 150*25 mm*10 um;mobile phase: [water (0.05% ammonia hydroxide v/v)−ACN]; B %: 39%-39%,12 min) to give A-75 (4.00 mg, 9.04 μmol, 1.6% yield, 99.7% purity) as ayellow solid.

LCMS: RT=2.95 min, m/z 441.2 [M+H]⁺

¹H NMR (CDCl3, 400 MHz) δ 8.21-8.12 (m, 2H), 7.61 (s, 2H), 4.90 (d,J=8.4 Hz, 1H), 3.91 (s, 3H), 3.85-3.80 (m, 3H), 3.06-3.05 (m, 2H), 2.70(m, 1H), 2.19-2.07 (m, 4H), 1.42-1.23 (m, 4H), 1.09 (m, 1H), 0.50-0.45(m, 2H), 0.18-0.14 (m, 2H).

Preparation of(1R,4R)—N¹-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N⁴-(pyridin-3-ylmethyl)cyclohexane-1,4-diamine (A-76)

To a solution of A-51 (200.00 mg, 554.20 μmol, 1.00 eq) andpyridine-3-carbaldehyde (59.36 mg, 554.20 μmol, 52.07 uL, 1.00 eq) wereadded AcOH (33.28 mg, 554.20 μmol, 31.70 μL, 1.00 eq) and NaBH₃CN (69.65mg, 1.11 mmol, 2.00 eq). The mixture was stirred at 15° C. for 16 hours.The mixture was quenched with aqueous NaHCO₃ (1 mL) and concentrated.The residue was purified by prep-HPLC (column: Phenomenex Gemini 150*25mm*10 um; mobile phase: [water (0.05% ammonia hydroxide v/v)−ACN]; B %:40%-58%, 12 min) to give A-76 (100.00 mg, 219.09 μmol, 39.5% yield,98.8% purity) as a yellow solid.

LCMS: RT=2.381 min, m/z 452.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ8.58-8.51 (m, 2H), 7.69 (d, J=8.0 Hz, 1H), 7.27(s, 1H), 4.87 (d, J=8.0 Hz, 1H), 3.91 (s, 3H), 3.85-3.81 (m, 3H),3.07-3.06 (m, 2H), 2.56-2.51 (m, 1H), 2.16-2.01 (m, 4H), 1.30-1.22 (m,4H), 1.09 (m, 1H), 0.50-0.46 (m, 2H), 0.19-0.16 (m, 2H).

Preparation of (1R,4R)—N¹-((1H-pyrazol-4-yl)methyl)-N⁴-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A80)

To a solution of A-14 (200.00 mg, 612.67 μmol, 1.00 eq) and1H-pyrazole-4-carbaldehyde (58.87 mg, 612.67 μmol, 1.00 eq) were addedAcOH (36.79 mg, 612.67 μmol, 35.04 uL, 1.00 eq) and NaBH₃CN (77.00 mg,1.23 mmol, 2.00 eq). The mixture was stirred at 15° C. for 16 hours. Themixture was quenched with aqueous NaHCO₃ (1 mL) and concentrated. Theresidue was purified by prep-HPLC (basic condition) to give A-80 (20.00mg, 48.90 μmol, 8% yield, 99.4% purity) as a yellow solid.

LCMS: RT=2.418 min, m/z 407.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.17 (d, J=5.2 Hz, 1H), 7.83 (s, 1H), 7.58 (s,1H), 6.71 (d, J=6.2 Hz, 1H), 4.88 (d, J=7.6 Hz, 1H), 3.89 (s, 4H), 3.81(s, 2H), 3.21 (d, J=6.0 Hz, 2H), 2.61 (s, 1H), 2.21-2.03 (m, 4H),1.36-1.25 (m, 4H), 1.09 (s, 1H), 0.48-0.46 (m, 2H), 0.25-0.24 (m, 2H).

Preparation of(1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N⁴-(1-methyl-1H-pyrazol-4-yl)methyl)cyclohexane-1,4-diamine(A81)

To a solution of A-14 (200.00 mg, 612.67 μmol, 1.00 eq) and compound81_1 (67.46 mg, 612.67 μmol, 1.00 eq) were added AcOH (36.79 mg, 612.67μmol, 35.04 L, 1.00 eq) and NaBH₃CN (77.00 mg, 1.23 mmol, 2.00 eq). Themixture was stirred at 15° C. for 16 hours. The mixture was quenchedwith a.q NaHCO₃ (1 mL) and concentrated. The residue was purified byprep-HPLC (basic condition) to give A-81 (40.00 mg, 87.81 μmol, 14.3%yield, 92% purity) as a yellow solid.

LCMS: RT=2.469 min, m/z 421.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.17 (d, J=5.2 Hz, 1H), 7.82 (s, 1H), 7.57 (s,1H), 7.51 (s, 1H), 6.7 (d, J=6.2 Hz, 1H), 4.84 (d, J=7.6 Hz, 1H),3.89-3.85 (m, 8H), 3.19 (d, J=6.0 Hz, 2H), 2.73 (s, 1H), 2.25-2.12 (m,4H), 1.54 (m, 1H), 1.29-1.23 (m, 2H), 1.07 (s, 1H), 0.50-0.46 (m, 2H),0.26-0.24 (m, 2H).

Preparation of(1R,4R)—N¹-((1H-pyrazol-3-yl)methyl)-N⁴-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-82)

To a solution of A-51 (200.00 mg, 554.20 μmol, 1.00 eq) and1H-pyrazole-3-carbaldehyde (53.25 mg, 554.20 μmol, 1.00 eq) were addedAcOH (31.7 μL, 554.20 μmol, 1.00 eq) and NaBH₃CN (69.65 mg, 1.11 mmol,2.00 eq). The mixture was stirred at 15° C. for 16 hours. The mixturewas quenched with a.q NaHCO₃ (1 mL) and concentrated. The residue waspurified by prep-HPLC (basic condition) to give A-82 (35.00 mg, 79.28μmol, 14.3% yield, 99.9% purity) as a yellow solid.

LCMS: RT=2.867 min, m/z 441.2 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.20 (s, 1H), 8.13 (s, 1H), 7.53 (s, 1H), 6.22(s, 1H), 4.91 (d, J=7.6 Hz, 1H), 3.93 (s, 2H), 3.91 (s, 3H), 3.81 (s,1H), 3.49 (s, 1H), 3.07 (d, J=5.6 Hz, 2H), 2.56-2.53 (m, 1H), 2.16-2.02(m, 4H), 1.33-1.32 (m, 4H), 1.05 (s, 1H), 0.48-0.45 (m, 2H), 0.18-0.16(m, 2H).

Preparation of(1R,4R)—N¹-(1-(1H-pyrazol-4-yl)ethyl)-N⁴-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A83)

To a solution of A-51 (100.00 mg, 251.67 μmol, 1.00 eq, HCl), compound83_1 (27.71 mg, 251.67 μmol, 1.00 eq) were added TEA (503.34 μmol, 69.77uL, 2.00 eq) and Ti(i-PrO)₄ (149 μL, 503.34 μmol, 2.00 eq). The mixturewas stirred at 80° C. for 12 hours. Then NaBH₃CN (39.54 mg, 629.18 μmol,2.50 eq) was added. The resulting mixture was stirred at 15° C. for 4hours. The mixture was quenched with a.q NaHCO₃ (50 mL) and extractedwith DCM (50 mL*2). The organic layer was concentrated and purified byprep-HPLC (basic condition) to give A-83 (20.00 mg, 43.96 μmol, 17.5%yield) as a yellow solid.

LCMS: RT=1.897 m/z 455.2[M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.19 (s, 1H), 8.12 (s, 1H), 7.52 (s, 2H), 4.90(d, J=7.6 Hz, 1H), 4.05-4.00 (m, 1H), 3.91 (s, 3H), 3.78-3.76 (m, 1H),3.06 (d, J=5.6 Hz, 2H), 2.49-2.46 (m, 1H), 2.11-2.08 (m, 4H), 1.40-1.38(m, 3H), 1.25-1.19 (m, 4H), 1.05 (s, 1H), 0.47-0.44 (m, 2H), 0.16-0.15(m, 2H).

Preparation ofN-((1R,4R)-4-((5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide(A87)

To a solution of compound 87_1 (46.59 mg, 415.65 μmol, 1.00 eq) in DCM(4 mL) were added DIEA (181.5 μL, 1.04 mmol, 2.50 eq), EDCI (95.62 mg,498.78 μmol, 1.20 eq) and HOBt (11.23 mg, 83.13 μmol, 0.20 eq) at 0° C.The reaction mixture was stirred at 0° C. for 0.5 hour. The solution ofA-51 (150.00 mg, 415.65 μmol, 1.00 eq) in DCM (4.5 mL) was added. Theresulting mixture was stirred at 20° C. for 16 hours. The mixture wasdiluted with H₂O (50 mL) and extracted with DCM (40 mL*3). The organiclayer was concentrated and purified by prep-HPLC (HCl condition) to giveA-87 (80.00 mg, 160.61 μmol, 38.6% yield, 98.6% purity, HCl) as a yellowsolid.

LCMS: RT=2.391 m/z 455.2[M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.48 (s, 1H), 8.39 (s, 1H), 8.16 (s, 2H), 4.04(s, 1H), 3.96 (s, 3H), 3.93-3.90 (m, 1H), 3.24 (d, J=6.4 Hz, 2H),2.18-2.04 (m, 4H), 1.67-1.53 (m, 4H), 1.18 (s, 1H), 0.57-0.55 (m, 2H),0.30-0.29 (m, 2H).

Preparation of(1R,4R)—N-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N⁴-((5-methyl-1H-pyrazol-4-yl)methyl)cyclohexane-1,4-diamine(A-91)

To a solution of A-51 (150.00 mg, 415.65 μmol, 1.00 eq) and5-methyl-1H-pyrazole-4-carbaldehyde (54.92 mg, 498.78 μmol, 1.20 eq) inMeOH (1.5 mL) were added AcOH (23.77 μL, 415.65 μmol, 1.00 eq) andNaBH₃CN (78.36 mg, 1.25 mmol, 3.00 eq). The mixture was stirred at 15°C. for 16 hours. The mixture was quenched with a.q NaHCO₃ (10 mL) andextracted with EA (20 mL*2). The combined organic layers wereconcentrated and purified by prep-HPLC (HCl condition) to give A-91(20.00 mg, 36.22 μmol, 8.7% yield, 89% purity, HCl) as a white solid.

LCMS: RT=2.425 m/z 455.2[M+H]⁺

¹HNMR (MeOD, 400 MHz) δ 8.44-8.41 (m, 2H), 8.23 (s, 1H), 4.26 (s, 2H),4.13-4.06 (m, 1H), 3.95 (s, 3H), 3.24 (d, J=6.4 Hz, 2H), 2.51 (s, 3H),2.40-2.24 (m, 4H), 1.76-1.61 (m, 4H), 1.09 (s, 1H), 0.54-0.53 (m, 2H),0.28-0.27 (m, 2H).

Preparation of(1R,4R)—N¹-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N⁴-(2,2,2-trifluoroethyl)cyclohexane-1,4-diamine(A94)

To a solution of A-51 (150.00 mg, 415.65 μmol, 1.00 eq) in THF (4.5 mL)were added TEA (144.04 μL, 1.04 mmol, 2.50 eq) and 2,2,2-trifluoroethyltrifluoromethanesulfonate (115.77 mg, 498.78 μmol, 1.20 eq). The mixturewas stirred at 50° C. for 16 hours. The mixture was concentrated andpurified by prep-HPLC (HCl condition) to give A-94 (80.00 mg, 163.38μmol, 39.3% yield, 97.9% purity, HCl) as a yellow solid.

LCMS: RT=2.137 min, m/z 443.2 [M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.45 (m, 2H), 4.15-4.09 (m, 2H), 4.09 (s, 1H),3.95 (s, 3H), 3.39-3.36 (m, 1H), 3.22-3.21 (m, 2H), 2.33-2.25 (m, 4H),1.71-1.56 (m, 4H), 1.09 (s, 1H), 0.54-0.52 (m, 2H), 0.28-0.27 (m, 2H).

Preparation of Preparation of(1R,4R)—N1-((1H-pyrazol-4-ylmethyl)-N4-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)-N1-(2,2,2-trifluoroethyl)cyclohexane-1,4-diamine(A-95)

To a solution of A-75 (150.00 mg, 340.16 μmol, 1.00 eq) in MeCN (4.5 mL)were added Cs₂CO₃ (221.66 mg, 680.32 μmol, 2.00 eq) and2,2,2-trifluoroethyl trifluoromethanesulfonate (118.43 mg, 510.24 μmol,1.50 eq). The mixture was stirred at 70° C. for 16 hours. The mixturewas filtered; the filtrate was concentrated and purified by prep-HPLC(HCl condition) to give A-95 (20.00 mg, 35.7 μmol, 10.5% yield, 100%purity, HCl) as a white solid.

LCMS: RT=2.285 m/z 523.2[M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.41-8.39 (m, 2H), 8.02 (s, 1H), 7.76 (s, 1H),5.03-4.97 (m, 2H), 4.23 (s, 2H), 4.12-4.04 (m, 1H), 3.95 (s, 3H), 3.21(d, J=6.4 Hz, 3H), 2.34-2.23 (m, 4H), 1.65-1.53 (m, 4H), 1.09 (s, 1H),0.54-0.52 (m, 2H), 0.27-0.26 (m, 2H).

Preparation of(1R,4R)—N¹,N¹-bis((1H-pyrazol-4-yl)methyl)-N⁴-(5-chloro-4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclohexane-1,4-diamine(A-96)

To a solution of A-75 (50.00 mg, 113.4 μmol, 1.00 eq) and compound1H-pyrazole-4-carbaldehyde (32.69 mg, 340.17 μmol, 3.00 eq) were addedTEA (31.44 μL, 226.78 μmol, 2.00 eq) and Ti(Oi-Pr)₄ (67.1 μL, 226.78μmol, 2.00 eq). The mixture was stirred at 80° C. for 12 hours. NaBH₃CN(21.38 mg, 340.17 μmol, 3.00 eq) was added and the resulting mixture wasstirred at 15° C. for 4 hours. The reaction was quenched with a.q NaHCO₃(1 mL) and filtered. The filtrate was purified by prep-HPLC (TFA andthen basic condition) to give A-%(7.00 mg, 13.10 umol, 11.6% yield,97.5% purity) as a white solid.

LCMS: RT=2.382 m/z 521.3[M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.23 (m, 2H), 8.03 (s, 1H), 7.78 (s, 4H),4.47-4.29 (m, 4H), 3.91-3.87 (m, 4H), 3.12 (d, J=6.4 Hz, 2H), 2.27-2.22(m, 4H), 1.94-1.85 (m, 2H), 1.46-1.36 (m, 2H), 1.01-1.00 (m, 1H),0.45-0.44 (m, 2H), 0.14-0.13 (m, 2H).

Preparation of(1R,4R)—N¹-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)-5-fluoropyrimidin-2-yl)cyclohexane-1,4-diamine(A-86

Step 1: To a solution of compound 517 (800 mg, 3.05 mmol, 1.00 eq) inDME (16 mL) were added compound 2 (509.54 mg, 3.05 mmol, 1.00 eq),Na₂CO₃ (2 M, 4.6 mL, 3.00 eq) and4-ditert-butylphosphanyl-N,N-dimethyl-aniline; dichloropalladium (108.04mg, 152.50 μmol, 108 uL, 0.05 eq) under nitrogen. The resulting mixturewas stirred at 85° C. for 2 hours under nitrogen. The mixture wasdiluted with H₂O (50 mL) and extracted with EA (50 mL). The organiclayer was concentrated to give compound 86_1 (1.20 g, crude) as a yellowoil.

LCMS: RT=1.819 min, m/z 267.1 [M+H]⁺

Step 2: The mixture of compound 86_1 (1.10 g, 4.12 mmol, 1.00 eq) andcompound 4 (1.88 g, 16.48 mmol, 4.00 eq) in dioxane (16 mL) was stirredat 130° C. for 8 hours under microwave. The mixture was filtered andconcentrated. The residue was purified by pre-HPLC (HCl condition) togive A-86 (1.30 g, crude) as a yellow solid. A-86 (300 mg) was purifiedagain by prep-HPLC (HCl condition) to give A-86 (20.00 mg, 52.51 μmol,12.1% yield, 100% purity, HCl) as a yellow solid.

LCMS: RT=2.321 min, m/z 345.2 [M+H]⁺

¹H NMR (MeOD, 400 MHz) δ 8.37-8.36 (m, 1H), 8.23-8.22 (m, 1H), 4.07-4.03(m, 1H), 3.96 (s, 3H), 3.21 (d, J=6.4 Hz, 2H), 2.24-2.18 (m, 4H),1.68-1.57 (m, 2H), 1.19 (s, 1H), 0.57-0.55 (m, 2H), 0.38-0.34 (m, 2H).

Preparation of(1R,4R)—N¹-((1H-pyrazol-4-yl)methyl)-N⁴-(4-(5-(cyclopropylmethyl)-1-methyl-1H-pyrazol-4-yl)-5-fluoropyrimidin-2-yl)cyclohexane-1,4-diamine(A-85)

To a solution of A-86 (300.00 mg, 783.90 μmol, 1.00 eq) and1H-pyrazole-4-carbaldehyde (82.86 mg, 862.29 μmol, 1.10 eq) in MeOH (3mL) were added AcOH (49.31 μL, 862.29 μmol, 1.10 eq) and NaBH₃CN (98.52mg, 1.57 mmol, 2.00 eq). The mixture was stirred at 15° C. for 16 hours.The mixture was quenched with a.q NaHCO₃ (1 mL) and concentrated. Theresidue product was purified by prep-HPLC (basic condition) to give A-85(50.00 mg, 111.53 μmol, 14.2% yield, 94.7% purity) as a yellow solid.

LCMS: RT=2.472 min, m/z 425.3 [M+H]⁺

¹H NMR (CDCl₃, 400 MHz) δ 8.10-8.09 (m, 1H), 8.02-8.01 (m, 1H), 7.60 (s,1H), 4.80 (d, J=8.4 Hz, 1H), 3.91 (s, 3H), 3.84 (m, 3H), 3.22-3.21 (m,2H), 2.68 (s, 1H), 2.21-2.06 (m, 4H), 1.41-1.35 (m, 2H), 1.30-1.24 (m,3H), 1.10-1.09 (m, 1H), 0.49-0.45 (m, 2H), 0.24-0.22 (m, 2H).

Biological Experimental Procedures

Primary Screen in RKO cells for compounds having typical CKIα inhibitoryactivity (β-catenin and p53 stabilization and histone H2AXphosphorylation; see Elyada et al, Nature 2011 Feb. 17;470(7334):409-13; Pribluda et al, Cancer Cell 2013 Aug. 12;24(2):242-56). RKO colorectal cells were incubated with 10 μM of each ofthe compounds for 16 hours at 37° C. Cells were washed with PBS and cellpellets were incubated with ice cold protein lysis buffer containingprotease inhibitor cocktail (1/200; Calbiochem) and phosphataseinhibitors (20 mM p-nitrophenyl phosphate (PNPP), 20 mMβ-glycerophosphate and 300 nM okadaic acid). Western blot analysis wasperformed by means of standard techniques. Blots were incubated withantibodies detecting β-catenin (1/2,500; BD Transduction), p53(DO-1&1801 hybridoma mix; dilution of 1:20 of supernatants from each),CKIα (C-19; 1/1,000; Santa Cruz Biotechnology) and phospho-histone H2AX(S139; 1/1,000; Millipore). Secondary antibodies were HRP-linked goatanti-mouse, goat anti-rabbit and rabbit anti-goat antibodies (all1/10,000; Jackson). Blots were developed using ECL (GE Healthcare).

Dose Response assay of the most active compounds. Active compounds werefurther analyzed in a dose-response experiment (FIGS. 1A and 1B).Similar to the primary screen, RKO cells were incubated with descendingconcentrations of each of the active compounds for 16 hours at 37° C.Cell extract isolation and Western Blot analysis were similar to theprimary screen.

Generation of a mouse model of CML blast crisis and inhibitor studies inthis model. To generate the BCR-ABL-inducible chronic myeloid leukemia(CML) model, bone marrow (BM) cells from 10 weeks wild type mouse wereextracted and enriched for cKit expressing cells (EasySep #18757) andincubated overnight at 37° C. in RPMI growth medium supplemented with15% FCS, L-Glutamine, Pen/Strep (Biological Industries, Israel) and stemcell factor (SCF), IL-3, IL-6 and TPO (Peprotech). The culture was theninfected with p210BCR-ABL-IRES-GFP retrovirus construct containingsupernatant medium for 4 hours, then growth medium was added andinfected cells were incubated at 37° C. for additional 24 h. The culturewas then injected I.V. into sublethally irradiated (500 rad) mice. Uponobserving a fast steady increase of GFP-expressing cells in theperipheral blood of inoculated mice (by FACS) and rising numbers ofleukocyte and immature cells (detected by Wright-Giemsa stained bloodfilms), mice were sacrificed and their BM cells were transferred to newsublethally irradiated WT hosts; each such transfer was termed diseasegeneration. By the fourth transfer, the hosts were no longer sublethallyirradiated prior to disease transfer. Blast crisis development wasreadily detectable by a highly abnormal number of blast cells, more than30% of white blood cells (WBC) in the peripheral blood (PB), and shorttime intervals between transfers. CKI inhibitor studies were performedon late generation diseases, in which PB blasts were easily detectable,with no host irradiation and a short generation time (up to 12 days).Mice were monitored daily for cachexia, weight loss, lethargy, and ruffcoats, and moribund mice were sacrificed upon moribund.

For evaluating CKIα inhibition effect on CML, a selective CKIα inhibitor(A14) was administered by oral gavage once a day at a dose of 10 mg/kg,starting from 24 h after BM transplantation (BMT) (FIG. 3A). Theinhibitor was dissolved in 1% methyl cellulose with 0.1% Tween 80 and0.2% Poly-ethylene glycol (Vehicle). Control mice were treated with thevehicle only.

Ex vivo inhibitor effects (FIGS. 2A and 13A-E). Freshly isolated BM fromAML (13A-E) or CML blast crisis (2A) carrying mice were grown in RPMIsupplemented with 15% FCS, L-Glutamine, Pen/Strep, Hepes, SodiumPyruvate and non-essential amino acids (Biological Industries, Israel).CKI inhibitors (A14, A51, A75 or A86) were dissolved in DMSO and addedto the tissue culture medium at the indicated concentrations; controlcultures were treated with DMSO only. Several hours following treatment(as indicated in each experiment), cells were harvested and countedmanually using a camera and standard inverted light microscope. Deadcells were excluded using Trypan Blue (Sigma). AnnexinV-PE (MBL), 7AAD(Tonbo) and PD-L1 (BioLegend) staining was evaluated by FACS accordingto manufacturer's recommendation.

Generation of a mouse model of acute myeloid leukemia (AML) (FIG. 6) andtreatment with the CKIα inhibitor A14 (FIGS. 7, 8 and 9) or A51 (FIGS.11 and 12). To generate the MLL-AF9 Acute myeloid leukemia (AML) model,bone marrow (BM) cells from 10 week old wild type mouse were extractedand enriched for cKit expressing cells (EasySep #18757) and incubatedovernight in RPMI supplemented with 15% FCS L-Glutamine, Pen/Strep(Biological Industries, Israel) and stem cell factor (SCF), IL-3, IL-6and TPO (Peprotech). The culture was infected with MSCV-MLL-AF9-IRES-GFPretrovirus construct containing supernatant medium for 4 hours, thengrowth medium was added and infected cells were incubated at 37° C. foradditional 24 h. The culture was then injected I.V. into sub lethallyirradiated (500 rad) mice. Upon detectable steady increase of GFPexpressing cells in the mice peripheral blood (checked by FACS analysis)and rise in leukocyte numbers and immature cells (detected byWright-Giemsa stained blood films), mice were sacrificed and their BMwas transferred (1^(st) BMT) to sublethally irradiated WT hosts. Uponemergence of AML disease mice were sacrificed and 50,000 BM cells weretransplanted (2^(nd) BMT) into WT host mice. GFP expressing cells weremonitored in the peripheral blood and upon detecting >10% GFP⁺ in PB(day 11 after BMT) mice were treated with A14 inhibitor (FIG. 7A). A14was administered by oral gavage once a day at a dose of 20 mg/kg for 3days followed by 10 mg/kg/day for 6 more days. The inhibitor wasdissolved in 1% methyl cellulose with 0.1% Tween 80 and 0.2%Poly-ethylene glycol (Vehicle). Control mice were treated with thevehicle only. Mice were monitored daily for cachexia, lethargy, and ruffcoats, and moribund mice were sacrificed. For a single dose experiment(FIG. 1A) A51 was administered by oral gavage at a single dose of 20mg/kg and mice were sacrificed 16 hours following treatment.

FACS analysis. All assays were performed on BD's equipment: FACScaliber, FACS ARIA sorter or LSR II machines. For immunostaining, cellswere suspended in a 1% BSA/PBS buffer with 5 μM EDTA. Cells were thenanalyzed by using Annexin V PE Apootosis Detection Kit (eBioscience),7-AAD (TONBO biosciences) and PE anti-mouse CD274 (B7-H1, PD-L1)antibody (clone 10F.9G2, BioLegend); Assays were performed according tothe manufacturer's instruction. Monoclonal antibodies specific for CD16and CD32 (Miltenyi Biotec) were used for blockade of Fc receptors beforestaining.

Complete blood counts. Peripheral venous blood was obtained from themouse facial vein using standard techniques and analyzed using the autohematology analyzer BC-2800 (Mindray) per manufacturer's instructions.

Table 1 provides quantified information for compounds of the inventionin activation of p53 and DNA damage response (DDR) and β-cateninstabilization as an indicator of Wnt pathway activation. p53 activationwas determined according to the degree of protein stabilization inseveral Western blot assays (Western blot examples are shown in FIGS. 1Aand 1B). For example, A43 stabilized p53 significantly above thenon-treatment control at 6 μM with no activity at 2 μM (FIG. 1A, lowerright panel) and thus received an average value of + for p53 activation.In contrast, A35 started stabilizing p53 at 0.2 μM, with maximalstabilization at 1 μM (FIG. 1A, upper right panel) and thus received anaverage value of +++ for p53 activation. A19-4, neither stabilized p53,nor induced γH2AX (a DNA damage response [DDR] indicator), butstabilized β-catenin at 2 μM, similarly to the best β-cateninstabilizing compounds (FIG. 1A, lower left panel) and thus received avalue of +++ for β-catenin/Wnt activation.

TABLE 1 p53, DNA damage response and Wnt/β-catenin activation ofcompounds of the invention DDR β- Com- Mass & p53 Catenin poundStructure Name (M + H⁺) activity activity A14

(1R,4R)-N¹-(4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₁₈H₂₇N₆: 327.2;Found: 327.2 +++ ++ A29-1

N-((1R,4R)-4- ((4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)amino) cyclohexyl)-2- methoxyacetamide Calc'd forC₂₁H₃₁N₆O₂: 399.2; Found: 399.2 ++ NA A27

(1R,4R)-N¹-(4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-N⁴- methylcyclohexane- 1,4-diamine Calc'd forC₁₉H₂₉N₆: 341.2.; Found: 341.2 ++ ++ A28

(1R,4R)-N¹-(4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-N⁴,N⁴- dimethylcyclohexane- 1,4-diamine Calc'd forC₂₀H₃₁N₆: 355.3; Found: 355.2 ++ +++ A36

(1R,4R)-N¹-(4- (1-cyclopentyl- 5- (cyclopropylmethyl)- 1H- pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₂₂H₃₃N₆: 381.3;Found: 381.4 ++ NA A39

(1R,4R)-N¹-(4- (5- (cyclopropylmethyl)- 1- (tetrahydro-2H-pyran-4-yl)-1H- pyrazol-4- yl)pyrimidin-2- yl)cyclohexane- 1,4-diamineCalc'd for C₂₂H₃₃N₆O: 397.3; Found: 397.4 ++ NA A29

(1R,4R)-N¹-(4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-N⁴-(2- methoxyethyl) cyclohexane-1,4- diamine Calc'dfor C₂₁H₃₃N₆O: 385.3; Found: 385.2 ++ NA A19-4

8-((4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)amino)-1,3- diazaspiro[4.5] decane-2,4-dione Calc'd for C₂₀H₂₆N₇O₂:396.2; Found: 396.2 NA +++ A35

(1R,4R)-N1-(4- (5- (cyclopropylmet hyl)-1- isopropyl-1H- pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₂₀H₃₁N₆: 355.3;Found: 355.3 +++ ++ A41

(1r,4S)-N1-(4- (5- (cyclopropylmethyl)- 1-((S)- tetrahydrofuran-3-yl)-1H- pyrazol-4- yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'dfor C₂₁H₃₁N₆O: 383.3; Found: 383.3 ++ NA A42

(1R,4R)-N1-(4- (5- (cyclopropylmethyl)- 1-((R)- tetrahydrofuran-3-yl)-1H- pyrazol-4- yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'dfor C₂₁H₃₁N₆O: 383.3; Found: 383.2 ++ NA A43

(1R,4R)-N1-(4- (5- (cyclopropylmethyl)- 1-(oxetan- 3-yl)-1H- pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₂₀H₂₉N₆O: 369.2;Found: 369.3 + NA A46

(1R,4R)-N1-(4- (5- (cyclopentylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₂₀H₃₁N₆: 355.3;Found: 355.2 ++ NA A38

(1R,4R)-N1-(4- (5- (cyclopropylmethyl)- 1- (tetrahydro-2H-pyran-3-yl)-1H- pyrazol-4- yl)pyrimidin-2- yl)cyclohexane- 1,4-diamineCalc'd for C₂₂H₃₃N₆O: 397.3; Found: 397.3 + + A45

(1R,4R)-N1-(4- (5- (cyclobutylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₁₉H₂₉N₆: 341.2;Found: 341.3 ++ + A19

(1-amino-4-((4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)amino) cyclohexyl) methanol Calc'd for C₁₉H₂₉N₆O:357.2; Found: 357.2 + + A26

8-((4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)amino)-3- oxa-1- azaspiro[4.5]decan- 2-one Calc'd for C₂₀H₂₇N₆O₂:383.2; Found: 383.2 + + A47

4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)-N-((1R,4R)-4-(piperidin-1- yl)cyclohexyl) pyrimidin-2- amine Calc'd for C₂₃H₃₅N₆:395.3; Found: 395.2 ++ + A48

4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)-N-((1R,4R)- 4-morpholinocyclohexyl) pyrimidin-2-amine Calc'd for C₂₂H₃₃N₆O: 397.3;Found: 397.3 + + A49

4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)-N-((1R,4R)-4-(pyrrolidin-1- yl)cyclohexyl) pyrimidin-2- amine Calc'd for C₂₂H₃₃N₆:381.3; Found: 381.3 + NA A50

N-((1R,4R)-4- (azetidin-1- yl)cyclohexyl)- 4-(5- (cyclopropylmethyl)-1-methyl- 1H-pyrazol-4- yl)pyrimidin-2- amine Calc'd for C₂₁H₃₁N₆:367.3; Found: 367.3 +++ ++ A51

(1R,4R)-N1-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₁₈H₂₆ClN₆:361.2; Found: 361.1 ++++ +++ A52

(1R,4R)-N1-(4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)-5-methylpyrimidin- 2- yl)cyclohexane- 1,4-diamine Calc'd for C₁₉H₂₉N₆:341.2; Found: 341.2 ++ NA A53

(1R,4R)-N1-(4- (5- (cyclobutylmethyl)- 1-isopropyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd for C₂₁H₃₃N₆: 369.3;Found: 369.2 + + A58

(4-(2-(((1R,4R)- 4- aminocyclohexyl) amino)pyrimidin- 4-yl)-1-methyl-1H- pyrazol-5- yl)(cyclopropyl) methanol Calc'd for C₁₈H₂₇N₆O:343.2; Found: 343.2 + NA A59

(1R,4R)-N¹-(5- chloro-4-(5- (cyclopropylmethyl)- 1- isopropyl-1H-pyrazol-4- yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd forC₂₀H₃₀ClN₆: 389.2; Found: 389.2 +++ ++ A56

(1R,4R)-N¹-(4- (1-methyl-5-((1- methylcyclopropyl) methyl)-1H- pyrazol-4-yl)pyrimidin- 2- yl)cyclohexane- 1,4-diamine Calc'd for C₁₉H₂₉N₆:341.2; Found: 341.2 ++ NA A57

(1R,4R)-N¹-(4- (1-methyl-5- neopentyl-1H- pyrazol-4- yl)pyrimidin-2-yl)cyclohexane- 1,4-diamine Calc'd for C₁₉H₃₁N₆: 343.3; Found: 343.2 NANA A30-1

(1R,4R)-N¹-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-4- methylcyclohexane- 1,4-diamine Calc'd forC₁₉H₂₈ClN₆: 375.2; Found: 375.2 ++++ ++ A30-2

(1s,4s)-N1-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-4- methylcyclohexane- 1,4-diamine Calc'd forC₁₉H₂₈ClN₆: 375.2; Found: 375.2 ++ ++ A60

(1R,4R)-N¹-(4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)-5-(trifluoromethyl) pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd forC₁₉H₂₆F₃N₆: 395.2; Found: 395.2 ++ +/− A64

N-((1R,4R)-4- (1H-pyrazol-1- yl)cyclohexyl)- 5-chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2- amineCalc'd for C₂₁H₂₇ClN₇: 412.2; Found: 412.2 + ++ A65

N-((1R,4R)-4- (1H-imidazol-1- yl)cyclohexyl)- 5-chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2- amineCalc'd for C₂₁H₂₇ClN₇: 412.2; Found: 412.2 ++ ++ A68

(1R,4R)-N¹-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-N⁴- phenylcyclohexane- 1,4-diamine Calc'd forC₂₄H₃₀ClN₆: 437.2; Found: 437.2 + + A71

(5r,8r)-8-((5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)amino)-1- azaspiro[4.5]decan- 2-one Calc'd forC₂₁H₂₈Cl N₆O: 415.2; Found: 415.2 NA ++ A74

(1R,4R)-N¹- benzyl-N⁴-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl-1H-pyrazol-4- yl)pyrimidin-2- yl)cyclohexane- 1,4-diamine Calc'd forC₂₅H₃₂ClN₆: 451.2; Found: 451.2 +++ ++ A75

(1R,4R)-N¹- ((1H-pyrazol-4- yl)methyl)-N⁴- (5-chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)cyclohexane- 1,4-diamine Calc'd for C₂₂H₃₀ClN₈: 441.2; Found: 441.2+++++ +++ A76

(1R,4R)-N¹-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-N⁴-(pyridin- 3- ylmethyl) cyclohexane- 1,4-diamineCalc'd for C₂₄H₃₁ClN₇: 452.2; Found: 452.2 +++ ++ A80

(1r,4r)-N¹- ((1H-pyrazol-4- yl)methyl)-N⁴- (4-(5- (cyclopropylmethyl)-1-methyl- 1H-pyrazol-4- yl)pyrimidin-2- yl)cyclohexane- 1,4-diamineCalc'd for C₂₂H₃₁N₈: 407.3; Found: 407.2 ++ NA A81

(1R,4R)-N¹- ((1H-pyrazol-3- yl)methyl)-N⁴- (5-chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)cyclohexane- 1,4-diamine Calc'd for C₂₃H₃₃N₈: 421.3; Found: 421.2 ++NA A82

(1R,4R)-N¹- ((1H-pyrazol-3- yl)methyl)-N⁴- (5-chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)cyclohexane- 1,4-diamine Calc'd for C₂₂H₃₀ClN₈: 441.2; Found: 441.2+++ ++ A83

(1R,4R)-N¹-(1- (1H-pyrazol-4- yl)ethyl)-N⁴-(5- chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)cyclohexane- 1,4-diamine Calc'd for C₂₃H₃₂ClN₈: 455.2; Found: 455.2++ ++ A87

N-((1R,4R)-4- ((5-chloro-4-(5- (cyclopropylmethyl)- 1-methyl-1H-pyrazol-4- yl)pyrimidin-2- yl)amino)cyclohexyl)- 1H- pyrazole-4-carboxamide Calc'd for C₂₂H₂₈Cl N₈O: 455.2; Found: 455.2 + +++ A91

(1R,4R)-N¹-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-N⁴-((5- methyl-1H- pyrazol-4- yl)methyl)cyclohexane-1,4- diamine Calc'd for C₂₃H₃₂ClN₈: 455.2; Found: 455.2 +++++ A94

(1R,4R)-N¹-(5- chloro-4-(5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4-yl)pyrimidin-2- yl)-N⁴-(2,2,2- trifluoroethyl) cyclohexane-1,4- diamineCalc'd for C₂₀H₂₇ClF₃N₆: 443.2; Found: 443.2 + ++ A95

(1R,4R)-N¹- ((1H-pyrazol-4- yl)methyl)-N⁴- (5-chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)-N¹-(2,2,2- trifluoroethyl) cyclohexane-1,4- diamine Calc'd forC₂₄H₃₁ClF₃N₆: 523.2; Found: 523.2 +++ ++ A96

(1R,4R)-N¹,N¹- bis((1H- pyrazol-4- yl)methyl)-N⁴- (5-chloro-4-(5-(cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)pyrimidin-2-yl)cyclohexane- 1,4-diamine Calc'd for C₂₆H₃₄ClN₁₀: 521.3; Found: 521.3NA NA A86

(1R,4R)-N¹-(4- (5- (cyclopropylmethyl)- 1-methyl- 1H-pyrazol-4- yl)-5-fluoropyrimidin- 2- yl)cyclohexane- 1,4-diamine Calc'd for C₁₈H₂₆FN₆:345.2; Found: 345.2 +++++ +++ A85

(1R,4R)-N¹- (1H-pyrazol-4- yl)methyl)-N⁴- (4-(5- (cyclopropylmethyl)-1-methyl- 1H-pyrazol-4- yl)-5- fluoropyrimidin- 2- yl)cyclohexane-1,4-diamine Calc'd for C₂₂H₃₀FN₆: 425.3; Found: 425.3 +++ +++ +indicates low, yet significant activation at a compound concentration of6 μM; +++ indicates maximal β-catenin or p53 stabilization at >2 μM;++++ indicates maximal activation of p53 and DDR at >0.5 μM, +++++indicates maximal activation of p53 and DDR at >0.1 μM.

IRAK1 Inhibition with compounds of the invention. RKO cells wereincubated for 16 hours at 37° C. with the indicated concentrations ofcompounds of the invention A51 (1 μM) and A14 (2 μM) (FIG. 10). At theindicated time points, RKO were treated with TNFα (100 units/ml). Cellswere harvested and analyzed by Western blot. Blots were incubated withthe following antibodies: Phospho-IRAK1 (Thr209), (A1074,AssayBiothechnology; 1/1,000), Phospho-IKKα/β (Ser176/180) (16A6, CellSignaling; 1/1,000), IKKα (2682, cell signaling; 1/1,1000), IKK β (2370,cell signaling; 1/1,1000), Phospho-c-Jun (Ser 63) (9261, cell signaling;1/1,1000), p53 (DO-1&1801 hybridoma mix; dilution of 1:20 ofsupernatants from each), CKIα (C-19; 1/1,000; Santa Cruz Biotechnology)and phospho-histoneH2AX (S139; 1/1,000; Millipore). Secondary antibodieswere HRP-linked goat anti-mouse, goat anti-rabbit and rabbit anti-goatantibodies (all 1/10,000; Jackson). Blots were developed using ECL (GEHealthcare). FIG. 10, shows the inhibition of phosphorylation of IRAK1as well as Phospho-IKKα/s and Phospho-c-Jun, indicative of IRAK1 kinaseInhibition. Also shown is p53 stabilization and phosphorylation of H2AX(γH2AX), a marker of DNA damage, indicative of CKIα kinase inhibition.CKIα protein levels serves as a loading control.

Kinome affinity scan for A51 (WXL5846, see Table 2 below), shows thatthe key targets of A51 includes the entire CKI family members and IRAK1,with a few control kinases. KINOMEscan™ is based on a competitionbinding assay that quantitatively measures the ability of a compound tocompete with an immobilized, active-site directed ligand. The assay isperformed by combining three components: DNA-tagged kinase; immobilizedligand; and a test compound. The ability of the test compound to competewith the immobilized ligand is measured via quantitative PCR of the DNAtag; % Ctrl=0 (zero) indicates a complete inhibition of the kinasetested by a concentration of 1 μM inhibitor (Fabian, M. A. et al. Asmall molecule-kinase interaction map for clinical kinase inhibitors.Nat. Biotechnol. 23, 329-336 (2005) and Karaman, M. W. et al. Aquantitative analysis of kinase inhibitor selectivity. Nat. Biotechnol.26, 127-132 (2008)).

TABLE 2 Kinome affinity scan for A51 (WXL5846) WXL05846 Target A51 GeneSymbol % Ctrl 1000 nM ERK1 94 ERK2 71 FAK 91 FGFR2 94 FGFR3 100 FLT3 89FLT3(ITD) 57 GSK3A 3.3 GSK3B 5 IGF1R 100 IKK-alpha 87 IKK-beta 77 INSR93 IRAK1 0 IRAK4 67 CSNK1A1 1.9 CSNK1A1L 0.5 CSNK1D 2.8 CSNK1E 0.1CSK1G2 0.7 CSNK2A1 22

Table 3 shows the Kd measurements for the interaction of A14 (WXL-4085)and A51 (WXL-5846) with IRAK1

TABLE 3 Kd of A14 (WXL-4085) and A51 (WXL-5846) with IRAK1 WXL-4085-02NWXL-5846 Target A14 A51 Gene Symbol Kd (nM) Kd (nM) IRAK1 11 2.9

Conclusion. IRAK1 as a superior target in a kinome scan shows zerobinding of IRAK1 to its target in the presence of the inhibitors.Pyrazole pyrimidine compounds of the invention A51 and A14 showedexcellent binding Kd to IRAK1. The compounds also showed inhibition ofIRAK1 activation and inhibition of the activation of the IRAK1 targetIKK (Ikappa B kinase) in RKO cells (Western blot analysis). It is to benoted that compound A51 showed complete (100%) inhibition of phospho-(active) IRAK1 at a concentration of 1 μM in the RKO cell line. As acomparison, Garrett W. Rhyasen et al. showed that Amgen's IRAK1-4inhibitor used in the treatment of MDS and breast cancer inhibited only70% of IRAK1/4 in cell lines at 10 μM (Garrett W. Rhyasen et al, 2013,Cancer Cell 24, 90-104, see especially FIG. 2 therein). Thus, compoundsof the invention, such as A51, are found to be excellent inhibitors ofIRAK1, an important upstream regulator of the NF-kB pathway which playsan important role in hematological malignancies (including among othersmultiple myeloma, MDS, leukemia and lymphoma, head and neck cancer andbreast cancer).

Single-dose treatment effect of a CKI inhibitor in AML mice and PD-L1expression. AML mice were prepared by inoculating MLL-AF9oncogene-transduced bone marrow cells to C57/BL6 mice. MLL-AF9 fusionrepresents one of a poor prognosis human AML induced by chromosomaltranslocation. 30 days after leukemia inoculation, the recipient micehave high white blood cells (WBC) counts (×10 higher than a normalmouse), and harbor>95% leukemia blasts in the bone marrow and 50% of theperipheral WBC in these mice are AML blasts. These mice havesplenomegaly and their bones are pale and fragile due to the acuteleukemia (FIG. 12). Oral treatment with A51 (20 mg/Kg) for 16 hrsresults in massive reduction of the total leukemia cells in the blood(FIG. 11B), shrinking of the leukemic spleen (FIG. 11C and FIG. 12A),50% and >90% reduction of proportion of leukemic blasts (GFP+ cells) inthe bone marrow and blood, respectively (FIG. 11D and FIG. 11E). Opaquebones turned to normal color following a single dose treatment (FIG.12B).

In vitro treatment effects of CKI inhibitors on AML cells isolated fromthe bone marrow of leukemic mice. Shown are the percentage of dead cells(7AAD+) following 10 or 100 nM inhibitor-treatment, at 6 and 9 hoursafter treatment (FIGS. 13B and 13D). DMSO treatment resulted in <10%dead cells at 9 hrs. Also, shown are effects of the inhibitors on theleukemia expression of the major immune checkpoint protein PD-L1 by flowcytometry analysis: reduction of the mean fluorescence intensity (MFI)at 5 hrs, and a decrease in the fraction of PD-L1-positive leukemiacells after inhibitor treatment in comparison to DMSO-treated cells at 6and 9 hours (decrease expressed by % of DMSO control) (FIGS. 13A, 13Cand 13E).

1. A compound having the general formula (I), including any stereoisomeror salt thereof:

wherein R₁ and R₂ are each independently selected from H, straight orbranched C₁-C₈ alkyl, straight or branched C₁-C₅ alkoxy, straight orbranched C₁-C₅ acyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl each optionallysubstituted by at least one of halide, hydroxyl, ester, ether, C₅-C₁₅aryl, C₃-C₇ heteroaryl, and amide; or R₁ and R₂ together with thenitrogen atom they are connected to form a 4-7 membered saturated,unsaturated or aromatic ring that may optionally include at least one ofN, O, NH, C═N, C═O or SO₂ and can optionally be substituted with atleast one of straight or branched C₁-C₅ alkyl, C₅-C₁₅ aryl, C₃-C₇heteroaryl, hydroxyl, halide and cyano; R₃ and R₄ are each independentlyselected from H, straight or branched C₁-C₈ alkyl optionally substitutedby at least one of halide, hydroxyl, alkoxy, C₅-C₁₅ aryl, C₃-C₇heteroaryl, ester and amide; or R₁ or R₂ together with R₃ and the carbonand nitrogen atom they are each connected to form a 4-7 memberedsaturated, unsaturated or aromatic ring that may optionally include atleast one of N, NH, O, C═N, C═O, SO₂, and can optionally be substitutedwith at least one of straight or branched C₁-C₅ alkyl, C₅-C₁₅ aryl,C₃-C₇ heteroaryl, hydroxyl, carbonyl, and halide; R₅ and R₈ are eachindependently selected from H, halide, straight or branched C₁-C₈ alkyl,straight or branched C₂-C₈ alkenyl, straight or branched C₂-C₈ alkynyl;optionally substituted by at least one halide; R₆ is selected fromstraight or branched C₁-C₈ alkyl, straight or branched C₂-C₈ alkenyl,straight or branched C₂-C₈ alkynyl, C₅-C₁₀ cycloalkyl, saturated orunsaturated 4-6 membered heterocyclyl; optionally substituted by atleast one of straight or branched C₁-C₈ alkyl, C₃-C₇ cycloalkyl, 4-6membered heterocyclyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl, halide, hydroxyl,C₁-C₅ alkyl halide; R₇ is selected from straight or branched C₁-C₈alkyl, straight or branched C₂-C₈ alkenyl, straight or branched C₂-C₈alkynyl; substituted by at least one C₃-C₇ cycloalkyl, 4-6 memberedheterocyclyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl, halide, hydroxyl, C₁-C₅alkyl halide.
 2. The compound according to claim 1, wherein R₁ and R₂are each independently selected from H, straight or branched C₁-C₈ alkyloptionally substituted by at least one of halide, C₅-C₁₅ aryl, C₃-C₇heteroaryl, hydroxyl, ester, ether and amide.
 3. The compound accordingto claim 1, wherein R₁ and R₂ are each independently selected from H,straight or branched C₁-C₅ alkoxy optionally substituted by at least oneof halide, hydroxyl, ester and amide.
 4. The compound according to claim1, wherein R₁ and R₂ are each independently selected from H, C₁-C₅ acyl,optionally substituted by at least one of halide, hydroxyl, ester, etherand amide.
 5. The compound according to claim 1, wherein R₁ and R₂ areeach independently selected from H, C₅-C₁₅ aryl, optionally substitutedby at least one of halide, hydroxyl, ester, ether and amide.
 6. Thecompound according to any one of the preceding claims, wherein R₄ is H.7. The compound according to any one of the preceding claims, wherein R₃and R₄ are H.
 8. The compound according to any one of the precedingclaims, wherein R₅ is selected from H, Cl and straight or branched C₁-C₄alkyl.
 9. The compound according to any one of the preceding claims,wherein R₅ is H.
 10. The compound according to any one of the precedingclaims, wherein R₈ is selected from H, Cl and straight or branched C₁-C₄alkyl
 11. The compound according to any one of the preceding claims,wherein R₈ is H.
 12. The compound according to any one of the precedingclaims, wherein one of R₅ or R₈ is H.
 13. The compound according to anyone of the preceding claims, wherein at least one of R₁ and R₂ is H. 14.The compound according to any one of the preceding claims, wherein R₆selected from straight or branched C₁-C₈ alkyl, C₅-C₁₀ cycloalkyl,saturated or unsaturated 4-6 membered heterocyclyl; and R₇ is selectedfrom straight or branched C₁-C₈ alkyl, substituted by at least one C₃-C₇cycloalkyl, 4-6 membered heterocyclyl, C₅-C₁₅ aryl, C₃-C₇ heteroaryl,halide, hydroxyl, C₁-C₅ alkyl halide.
 15. The compound according to anyone of the preceding claims, wherein R₆ is selected from a straight orbranched C₁-C₈ alkyl, C₅-C₁₀ cycloalkyl, 4-6 membered saturatedheterocyclyl.
 16. The compound according to any one of the precedingclaims, wherein R₇ is a straight or branched C₁-C₈ alkyl substituted byat least one of C₃-C₇ cycloalkyl and hydroxyl.
 17. The compoundaccording to any one of the preceding claims, wherein R₆ is selectedfrom straight or branched C₁-C₈ alkyl, saturated, unsaturated oraromatic 4-6 membered heterocyclyl, each optionally substituted by atleast one of straight or branched C₁-C₈ alkyl, C₃-C₇ cycloalkyl, halide,hydroxyl, CF₃.
 18. The compound according to any one of the precedingclaims, wherein R₇ is a straight or branched C₁-C₈ alkyl substituted byat least one C₃-C₇ cycloalkyl.
 19. The compound according to any one ofthe preceding claims, wherein R₁ and R₂ together with the nitrogen atomthey are connected to form a 4-7 membered saturated ring optionallyincluding at least one of N, O, NH, C═N, C═O or SO₂ and can optionallybe substituted with at least one of straight or branched C₁-C₅ alkyl,hydroxyl, halide and cyano.
 20. The compound according to any one of thepreceding claims, wherein R₁ and R₂ together with the nitrogen atom theyare connected to form a 4-7 membered saturated ring.
 21. The compoundaccording to any one of the preceding claims, wherein R₁ and R₂ togetherwith the nitrogen atom they are connected to form a 4-7 memberedsaturated ring including at least one of N or O.
 22. The compoundaccording to any one of the preceding claims, wherein R₁ and R₂ togetherwith the nitrogen atom they are connected to form a 4-7 memberedaromatic ring optionally including at least one of N or
 0. 23. Thecompound according to any one of the preceding claims, wherein R₃ and R₄are H.
 24. The compound according to any one of the preceding claims,wherein R₁ or R₂ together with R₃ and the carbon and nitrogen atom theyare connected to form a 4-7 membered saturated ring that optionallyincludes at least one of N, NH, O, C═O, SO₂, and can optionally besubstituted with at least one of straight or branched C₁-C₅ alkyl,hydroxyl, carbonyl, and halide.
 25. The compound according to any one ofthe preceding claims, wherein R₁ or R₂ together with R₃ and the carbonand nitrogen atom they are connected to form a 4-7 membered saturatedring that includes at least one of NH, O, C═O.
 26. A compound accordingto any one of the preceding claims, selected from the following:


27. A composition comprising at least one compound according to any oneof claims 1 to
 26. 28. A compound according to any one of claims 1 to26, for use in therapy.
 29. A compound according to any one of claims 1to 26, for use in the inhibition of and least one of Casein kinase I(CKI) and Interleukin-1 receptor-associated kinase 1 (IRAK1).
 30. Acompound according to any one of claims 1 to 26, for use in theinhibition of Casein kinase I (CKI).
 31. A compound according to any oneof claims 1 to 26, for use in the inhibition of Interleukin-1receptor-associated kinase 1 (IRAK1).
 32. A compound according to anyone of claims 1 to 26, for use in inducing anti-tumor response.
 33. Acompound for use according to claim 32, wherein said anti-tumor responsecomprises cancer immunotherapy response.
 34. A compound according to anyone of claims 1 to 26, for use in the treatment of a condition, symptomor disease associated with a malignant condition.
 35. A compound for useaccording claim 34, wherein said malignant condition is cancer.
 36. Acompound for use according claim 34, wherein said malignant condition isselected from hematological malignancies (Multiple Myeloma,Myelodysplastic Syndrome (MDS), Acute Myeloid Leukemia (AML), Melanomaand ER-negative breast cancer, Diffuse Large B cell lymphoma (DLBCL),Chronic Myelogenous Leukemia (CML), Chronic lymphocytic leukemia (CLL),head and neck cancer and any combinations thereof.
 37. A compound foruse according claim 35, wherein said cancer has WT p53.
 38. A compoundaccording to any one of claims 1 to 26, for use in the treatment ofcancer having WT p53, wherein said WT p53 is a biomarker for the saidcompound efficacy.
 39. A compound for use according claim 35, whereinsaid cancer is selected from Multiple myeloma, leukemia, malignantmelanoma, breast cancer, prostate cancer, colorectal cancer and anycombinations thereof.
 40. A compound for use according to any one ofclaims 34 to 39, further inducing cancer immunotherapy response.
 41. Acompound according to any one of claims 1 to 26, for use in thetreatment of an inflammatory and immune related disorder including acondition, symptom or disease associated therewith.
 42. A method ofinhibiting at least one of Casein kinase I (CKI) and Interleukin-1receptor-associated kinase 1 (IRAK1) in a subject in need thereofcomprising the step of administrating to said subject at least onecompound according to any one of claims 1 to
 26. 43. A method ofinhibiting Casein kinase I (CKI) in a subject in need thereof comprisingthe step of administrating to said subject at least one compoundaccording to any one of claims 1 to
 26. 44. A method of inhibitingInterleukin-1 receptor-associated kinase 1 (IRAK1) in a subject in needthereof comprising the step of administrating to said subject at leastone compound according to any one of claims 1 to
 26. 45. A method oftreating a condition, symptom or disease associated with a malignantcondition in a subject in need thereof, said method comprising the stepof administering to said subject at least one compound according to anyone of claims 1 to
 26. 46. A method according claim 45, wherein saidmalignant condition is cancer.
 47. A method according claim 46, whereinsaid cancer has WT p53.
 48. A method of treating cancer having WT p53 ina subject in need thereof, wherein said WT p53 is a biomarker for thesaid compound efficacy.
 49. A method according to claim 46, wherein saidcancer is selected from leukemia, multiple myeloma, malignant melanoma,breast cancer, prostate cancer and colorectal cancer.
 50. A methodaccording to claim 45, wherein said malignant condition is selected fromhematological malignancies (Multiple myeloma, Myelodysplastic Syndrome(MDS), Acute Myeloid Leukemia (AML), Melanoma and ER-negative breastcancer, Diffuse Large B cell lymphoma (DLBCL), Chronic MyelogenousLeukemia (CML), Chronic lymphocytic leukemia (CLL), head and neck cancerand any combinations thereof.
 51. A method according to any one ofclaims 42 to 50, further comprising inducing a cancer immunotherapyresponse in said subject.
 52. A method for inducing an immunotherapyresponse in a subject in need thereof, said method comprising the stepof administering to said subject at least one compound according to anyone of claims 1 to
 26. 53. A method of treating an inflammatory andimmune related disorder, including a condition, symptom or diseaseassociated therewith in a subject in need thereof, said methodcomprising the step of administering to said subject at least onecompound according to any one of claims 1 to 26.